Download - Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin resistance
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NEUTRALIZATION OF OSTEOPONTIN INHIBITS OBESITY-INDUCED INFLAMMATION AND INSULIN RESISTANCE
Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger, Birgit Pfau, Angelika Neuhofer, Thomas Weichhart, Marcus D. Saemann, Rene Geyeregger,1 Michaela Schlederer,3Lukas Kenner, and Thomas M. Stulnig
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WHAT IS OSTEOPONTIN (OPN)?Glycosylated phosphoprotein (SPP1
gene)Pro-inflammatory cytokineFunctions:
Bone formationInflammationCell migration
Implication in diseases
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OBESITY & INFLAMMATIONChronic low-grade inflammation Inflammation insulin resistance
metabolic diseases
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OBESITY, INFLAMMATION, OSTEOPONTIN
OPN
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OBJECTIVE OF STUDYOPN neutralizing antibodyHypothesis
Targeting OPN in obesity could reverse adipose tissue inflammation and insulin resistance
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MICE MODELS (C57BL/6) Induced Obesity 24 weeks High Fat
Diet (HFD)Normal Chow (NC)
HFD NC
ControlAntibody
OPN-neutralizing
Antibody
OPN-neutralizing
AntibodyControl
Antibody
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NEUTRALIZING ANTIBODY• Goat Anti-Mouse OPN-specific antibodies • Control Goat antibody
MOUSE OPN
Production of antibodies against mouse OPN
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OVERVIEW OF STUDY1. Insulin Sensitivity
Insulin Tolerance Test HOMA-IR
2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay
3. Adipose Tissue Inflammation Western Blot
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INSULIN SENSITIVITY: INSULIN TOLERANCE TESTAssess response to insulinFast 3 hrs blood glucose levels measured
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INSULIN SENSITIVITY: HOMA-IRHomeostasis Model Assessment of Insulin
Resistance HOMA-IR values insulin sensitivity high “ “ insulin resistance
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INSULIN SENSITIVITY OPN neutralization enhanced
insulin sensitivityITT
Anti-OPN group HF respond better to insulin
HOMA-IRIR significantly lower after anti-OPN treatment
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OVERVIEW OF STUDY1. Insulin Sensitivity
Insulin Tolerance Test HOMA-IR
2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay
3. Adipose Tissue Inflammation Western Blot
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MACROPHAGE INFILTRATION: RT-PCR SYBR GREEN
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MACROPHAGE INFILTRATION: IMMUNOFLUORESCENCE
F4/80
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MACROPHAGE INFILTRATION: FLOW CYTOMETRY
Peripheral Blood
Addition of antibody
Skin
Alignment of cells in single file
Cell Culture
Fluid Portion
Charge Assigner
AdiposeAdipose
Stromal VascularFraction
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MACROPHAGE INFILTRATION: FLOW CYTOMETRY
• SVC: Stromal Vascular cells• F4/80= macrophage marker• less macrophage infiltration in adipose tissue of anti-OPN group
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MACROPHAGE INFILTRATION: TUNEL ASSAYTerminal deoxynucleotidyl
transferase-mediated d-UTP Nick End Labeling
Apoptosis “nicks” in DNA
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MACROPHAGE INFILTRATION: TUNEL ASSAY
Enhanced apoptosis in anti-OPN-treated mice disappearance of macrophages
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MACROPHAGE ACCUMULATION IN ADIPOSE TISSUEOPN Neutralization inhibits
macrophage infiltrationRT-PCR
macrophage marker mRNAImmunofluorescence
lit areas on adipose tissueFlow Cytometry
% F4/80 + cells of SVCTUNEL
apoptosis (HF anti-OPN)
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OVERVIEW OF STUDY1. Insulin Sensitivity
Insulin Tolerance Test HOMA-IR
2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay
3. Adipose Tissue Inflammation Western Blot
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ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT
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ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT cJun NH2-terminal kinase (JNK) signaling
pathway required in macrophages to establish insulin resistance and inflammation
Phosphorylated JNK activated
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ADIPOSE TISSUE INFLAMMATION:
Anti-OPN treatment abolished JNK phosphorylation in obese but not lean mice
OPN neutralization effectively decreases deleterious inflammatory alterations in adipose tissue of obese mice
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SUMMARY OF FINDINGSAntibody-mediated OPN
neutralizationEnhanced insulin sensitivity Inhibited macrophage
accumulation in adipose tissue (apoptosis)
Attenuated adipose tissue inflammation
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BIG PICTURE Inflammation insulin resistance
metabolic diseasesOPN in obesity
Inflammation attract macrophages into adipocytes
OPN neutralization stop influx of macrophages less
inflammation better insulin sensitivity