Post on 22-Feb-2016
description
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
WHAT IS OSTEOPONTIN (OPN)?Glycosylated phosphoprotein (SPP1
gene)Pro-inflammatory cytokineFunctions:
Bone formationInflammationCell migration
Implication in diseases
OBESITY & INFLAMMATIONChronic low-grade inflammation Inflammation insulin resistance
metabolic diseases
OBESITY, INFLAMMATION, OSTEOPONTIN
OPN
OBJECTIVE OF STUDYOPN neutralizing antibodyHypothesis
Targeting OPN in obesity could reverse adipose tissue inflammation and insulin resistance
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
NEUTRALIZING ANTIBODY• Goat Anti-Mouse OPN-specific antibodies • Control Goat antibody
MOUSE OPN
Production of antibodies against mouse OPN
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
INSULIN SENSITIVITY: INSULIN TOLERANCE TESTAssess response to insulinFast 3 hrs blood glucose levels measured
INSULIN SENSITIVITY: HOMA-IRHomeostasis Model Assessment of Insulin
Resistance HOMA-IR values insulin sensitivity high “ “ insulin resistance
INSULIN SENSITIVITY OPN neutralization enhanced
insulin sensitivityITT
Anti-OPN group HF respond better to insulin
HOMA-IRIR significantly lower after anti-OPN treatment
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
MACROPHAGE INFILTRATION: RT-PCR SYBR GREEN
MACROPHAGE INFILTRATION: IMMUNOFLUORESCENCE
F4/80
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
MACROPHAGE INFILTRATION: FLOW CYTOMETRY
• SVC: Stromal Vascular cells• F4/80= macrophage marker• less macrophage infiltration in adipose tissue of anti-OPN group
MACROPHAGE INFILTRATION: TUNEL ASSAYTerminal deoxynucleotidyl
transferase-mediated d-UTP Nick End Labeling
Apoptosis “nicks” in DNA
MACROPHAGE INFILTRATION: TUNEL ASSAY
Enhanced apoptosis in anti-OPN-treated mice disappearance of macrophages
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)
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
ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT
ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT cJun NH2-terminal kinase (JNK) signaling
pathway required in macrophages to establish insulin resistance and inflammation
Phosphorylated JNK activated
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
SUMMARY OF FINDINGSAntibody-mediated OPN
neutralizationEnhanced insulin sensitivity Inhibited macrophage
accumulation in adipose tissue (apoptosis)
Attenuated adipose tissue inflammation
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