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