The blood-brain barrier transfer coefficient (Ki) transiently increases after infusion of TGRL lipolysis products. A: the mean Ki (min−1) + SE of C57BL/6 (WT, n = 4) and Ldlr−/− (n = 4) animals on a control diet are plotted at baseline (pretreatment), 20, 40, and 60 min postinfusion of TGRL lipolysis products (150 mg/dl TGRL + 2 U/ml LpL). Two-way repeated-measures ANOVA showed statistical significance for the effect of time (combined data; P ≤ 0.05, n = 8) and the Student-Newman-Keuls post hoc test demonstrated a significant difference (P ≤ 0.05, n = 8) for C567BL/6 and Ldlr−/−mice (pooled) at 0–20 min vs. each of the other time points. B: representative images of Ki maps for WT and Ldlr−/− mouse brains (circled in red) for baseline (pretreatment), 20, 40, and 60 min post-TGRL lipolysis product (TGRL + LpL) bolus infusion.
TGRL lipolysis products induce lipid droplets in normal human astrocyte. Normal human astrocytes were treated with media (M), LpL (L, 2 U/ml), TGRL (T, 150 mg/dl) or TGRL+ LpL (T+L) for 3 h. A: bright-field images of cells stained with oil red o and counterstained with hematoxylin show increased lipid droplet formation after lipolysis product exposure (arrows). B: number of lipid droplets per cell was determined from 3 separate experiments. Data are represented as means ± SE. *P ≤ 0.05 when compared with media (scale bar, 20 μm).
Stress and inflammatory-related gene transcript up- or downregulated by TGRL lipolysis products. Astrocytes were exposed to media, LpL (2 U/ml), TGRL (150 mg/dl), and TGRL + LpL for 3 h and quantitative RT-PCR was used to validate changes in gene transcription of top 10 upregulated genes from microarray (A), genes related to inflammation or lipid droplets (B), or downregulated genes (C). Values are normalized to GAPDH and change in expression is represented as fold change when compared with media. Data are represented as means ± SE. *P ≤ 0.05, when compared with media and n = 3.
Lipolysis products increase phosphorylation of JNK and cJUN as well as increase activating transcription factor 3 (ATF3) abundance. Normal human astrocytes were treated with media (M), LpL (L, 2 U/ml), TGRL (T, 150 mg/dl) or TGRL+ LpL (T + L) for 3 h. Cell lysates were prepared and subjected to Western blot analysis, and representative blots (a) and densitometry analysis (b) are shown. A–C: protein expression of p-JNK (A), p-cJUN (B), and ATF3 (C) after the 4 treatments described above. Densitometric analysis of ATF3 is relative to β-actin while p-JNK and p-cJUN are relative to total and β-actin. Data are represented as means ± SE. *P ≤ 0.05 when compared with media and n = 3.
TGRL lipolysis products increases nuclear accumulation C/EBP homologous protein (CHOP). Normal human astrocytes were treated with media (M), LpL (L, 2 U/ml), TGRL (T, 150 mg/dl), or TGRL+ LpL (T+L) for 3 h. A: immunofluorescence images of CHOP showing nuclear accumulation (red) and 4',6-diamidino-2-phenylindole (DAPI, blue) for nucleus stain. B: nuclear fluorescent intensity of CHOP immunostaining in arbitrary units (AU). C: percentage of cells with nuclear staining for CHOP after lipolysis product exposure, n = 5 coverslips/treatment group. Western blot analysis of CHOP after treatments listed above. Representative blots (D) and densitometry analysis relative to β-actin (E) are shown. Data are represented as means ± SE. *P ≤ 0.05 when compared with media (scale bar, 20 μm).
Lipolysis products increase phosphorylation of NF-κB p65 subunit and reduce IκBα, inhibitor of NF-κB. Normal human astrocytes were treated with media (M), LpL (L, 2 U/ml), TGRL (T, 150 mg/dl), or TGRL+ LpL (T + L) for 3 h. Cell lysates were prepared and subjected to Western blot analysis. Representative blots (a) and densitometry analysis (b) are shown. A and B: protein expression of p-p65 (A) and IκBα (B) after the 4 treatments described above. Densitometric analysis of p-p65 are relative to total and β-actin while IκBα is relative to β-actin. Data are represented as means ± SE. *P ≤ 0.05 when compared with media and n = at least 3.
Human astrocytes secrete IL-8, macrophage inflammatory protein-3α (MIP-3α), and growth differentiation factor-15 (GDF15)in response to lipolysis products from human TGRL. Astrocytes were treated for 3 h with media (M), LpL (L, 2 U/ml), TGRL (T, 150 mg/dl), or TGRL+ LpL (T + L) and then changed to fresh media and incubated for additional 21 h (24 h from start of treatment). Supernatants were subjected to ELISA for IL-8 (A), MIP-3α (B), and GDF-15 (C). Data are represented as means ± SE. *P ≤ 0.05, when compared with media and n = 3.
Human astrocytes secrete IL-8, MIP-3α, and IL-6 in response to stearic acid but not linoleic, palmitic or oleic acid. Astrocytes were treated for 3 h with media (M), ethanol (EtOH, control), or fatty acids palmitic (PA), stearic (SA), linoleic (LA), or oleic acid (OA) at 150 µM and then changed to fresh media and incubated for additional 21 h (24 h from start of treatment). Supernatants were subjected to ELISA for IL-8 (A), MIP-3α (B), and IL-6 (C). Data are represented as means ± SE. *P ≤ 0.05, when compared with media or EtOH where n = 3.
The oligonucleotide sequences for each primer sequence were obtained from Affymetrix database using the probe set IDs. The primers were custom prepared and used as described in materials and methods.
Summary of genome-wide responses of astrocytes to LpL, TGRL, or TGRL + LpL
Total No. of Genes Affected
%Total Genes Affected
TGRL + LpL
All data are based on over 13,000 genes reliably detected in astrocytes. The criterion for selection was (±) ≥2-fold change as detailed in materialsandmethods. Effects of lipoprotein lipase (LpL; 2 U/ml), triglyceride-rich lipoprotein (TGRL; 150 mg/dl), or TGRL + LpL on gene expression in normal human astrocytes (NHA) were obtained by comparing the entire list of genes from astrocytes grown in basal media. The number differentially expressed LpL, TGRL, or TGRL + LpL up (column 2)- or downregulated genes (column 3) are shown as the percentage of the total number of genes affected in the astrocytes (column 4).
Significantly enriched GO terms of the highly expressed genes in human astrocytes treated with LpL, TGRL, or TGRL + LpL
TGRL + LpL
Regulation of cell proliferation
Positive regulation of macromolecule metabolic process
Positive regulation of cellular biosynthetic process
Positive regulation of biosynthetic process
Positive regulation of cell proliferation
Positive regulation of macromolecule biosynthetic process
Positive regulation of nitrogen compound metabolic process
Negative regulation of cell proliferation
Regulation of transcription
Regulation of transcription from RNA polymerase II promoter
Growth factor activity
Regulation of programmed cell death
Regulation of cell death
Transcription factor activity
Positive regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
Regulation of apoptosis
Positive regulation of gene expression
Positive regulation of transcription
Positive regulation of RNA metabolic process
Regulation of transcription, DNA-dependent
Response to hypoxia
Regulation of RNA metabolic process
Transcription regulator activity
Programmed cell death
Response to oxygen levels
Response to bacterium
Positive regulation of transcription, DNA-dependent
Blood vessel development
Negative regulation of programmed cell death
Negative regulation of cell death
Response to molecule of bacterial origin
Positive regulation of transcription from RNA polymerase II promoter
Blood vessel morphogenesis
Negative regulation of apoptosis
Intracellular nonmembrane-bounded organelle
Positive regulation of cell division
Extracellular region part
Regulation of nervous system development
Response to extracellular stimulus
Localization of cell
Hair cycle process
Hair follicle development
Molting cycle process
Response to wounding
Negative regulation of transcription factor activity
Regulation of cell division
Transcription repressor activity
Negative regulation of cell differentiation
Negative regulation of multicellular organismal process
Regulation of phosphorylation
Negative regulation of DNA binding
Protein kinase cascade
Regulation of neurogenesis
Response to endogenous stimulus
Response to nutrient levels
Intracellular organelle lumen
Response to organic substance
Protein dimerization activity
Regulation of DNA binding
Intracellular signaling cascade
Regulation of phosphate metabolic process
Regulation of phosphorus metabolic process
Response to lipopolysaccharide
Regulation of cell development
Negative regulation of cellular biosynthetic process
Negative regulation of transcription
Regulation of binding
Regulation of transcription factor activity
Regulation of cell cycle
Negative regulation of binding
Positive regulation of cell-cell adhesion
Negative regulation of biosynthetic process
Regulation of gene-specific transcription
DAVID was selected to perform functional enrichment analysis to determine enrichment for Gene Ontology (GO) biological process (GO terms). Statistical significance was determined using one-tailed Fishers’ extract test and Benjamini correction; adjusted P ≤ 0.05 between two groups was considered significant.
Human astrocyte genes upregulated by TGRL lipolysis products
Genes shown to be downregulated in astrocytes by lipolysis product treatment are classified functionally into apoptosis/cell cycle, inflammatory responses, metabolism, transcription factors, ion metabolism, and protein binding classes.