Transcriptional control of intestinal cholesterol absorption, adipose energy expenditure and lipid handling by Sortilin

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr−/−) atherosclerosis model, Sortilin deficiency (Sort1−/−) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr−/−Sort1−/− mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease

A Novel Spectral Annotation Strategy Streamlines Reporting of mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4-hour exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method

Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance

Objectives: Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. Methodology/Principal Findings Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. Conclusion: ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance

Enforced expression of human ANGPTL2 increased adipose tissue macrophages and promoted M1 macrophage polarization in adipose tissue from db/db mice.

<p>A, Stromal vascular fraction (SVF) was isolated from the epididymal fat pad then stained with F4/80, CD11b, and CD11c antibodies and analyzed by FACS. B, Adipose tissue macrophage number was determined as F4/80+ and CD11b+ fraction and determined by FACS (n = 7–8). Data are mean ± SEM, **: P<0.01, *: P<0.05 compared with LacZ group.</p

Enforced expression of human ANGPTL2 impaired glucose and insulin tolerance in mice.

<p>A, Western blot analysis of FLAG-tagged ANGPTL2 in the liver (upper panel: lean mice, lower panel: db/db mice). B, Body weight (n = 7–8 mice / group). C, Plasma ANGPTL2 levels 2 week after adenovirus injection (n = 6–8). D, Fasting glucose levels at 2 weeks (n = 7–8 mice / group). E, Glucose tolerance test at 2 weeks after treatment (Left: Lean mice, Right: db/db mice, n = 7–8 mice / group, in Experiment 1). F, Insulin tolerance test 2 weeks after treatment in db/db mice (n = 7–8 mice / group, in Experiment 2). G, Quantitative RT-PCR of mRNAs encoding gluconeogenesis related genes in the liver (Left: Lean mice, Right: db/db mice, n = 8). Data are mean ± SEM, **: P<0.01, *: P<0.05 compared with LacZ group.</p

ANGPTL2 enhanced hepatic lipid accumulation in mice.

<p>A, Representative images of the liver (Left: LacZ, Right: Angptl2, db/db mice). B, Liver triglyceride levels (Left: Lean mice, Right: db/db mice, n = 7–8 animals per group). C, Representative images of oil red O staining (Left: LacZ, Right: Angptl2, db/db mice). D, Oil red O staining area (n = 7–8, db/db mice). E, Quantitative RT-PCR of mRNAs encoding genes related to fatty acid metabolism in the liver of lean mice (n = 8) F, Quantitative RT-PCR of mRNAs encoding genes related to fatty acid metabolism in the liver of db/db mice (n = 7–8). Data are mean ± SEM, **: P<0.01, *: P<0.05 compared with LacZ group.</p