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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,3 Lukas Kenner, and Thomas M. Stulnig

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Page 1: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

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

Page 2: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

WHAT IS OSTEOPONTIN (OPN)?

Glycosylated phosphoprotein (SPP1 gene)

Pro-inflammatory cytokineFunctions:

Bone formationInflammationCell migration

Implication in diseases

Page 3: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OBESITY & INFLAMMATIONChronic low-grade inflammation Inflammation insulin resistance

metabolic diseases

Page 4: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OBESITY, INFLAMMATION, OSTEOPONTIN

OPN

Page 5: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OBJECTIVE OF STUDY

OPN neutralizing antibodyHypothesis

Targeting OPN in obesity could reverse adipose tissue inflammation and insulin resistance

Page 6: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MICE MODELS (C57BL/6) Induced Obesity 24 weeks High Fat

Diet (HFD)Normal Chow (NC)

HFD NC

ControlAntibody

OPN-neutralizing

Antibody

OPN-neutralizing

Antibody

ControlAntibody

Page 7: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

NEUTRALIZING ANTIBODY

• Goat Anti-Mouse OPN-specific antibodies • Control Goat antibody

MOUSE OPN

Production of antibodies against mouse OPN

Page 8: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OVERVIEW OF STUDY

1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR

2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay

3. Adipose Tissue Inflammation Western Blot

Page 9: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

INSULIN SENSITIVITY: INSULIN TOLERANCE TESTAssess response to insulinFast 3 hrs blood glucose levels measured

Page 10: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

INSULIN SENSITIVITY: HOMA-IRHomeostasis Model Assessment of Insulin

Resistance HOMA-IR values insulin sensitivity high “ “ insulin resistance

Page 11: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

INSULIN SENSITIVITY

OPN neutralization enhanced insulin sensitivityITT

Anti-OPN group HF respond better to insulin

HOMA-IRIR significantly lower after anti-OPN treatment

Page 12: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OVERVIEW OF STUDY

1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR

2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay

3. Adipose Tissue Inflammation Western Blot

Page 13: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MACROPHAGE INFILTRATION: RT-PCR SYBR GREEN

Page 14: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MACROPHAGE INFILTRATION: IMMUNOFLUORESCENCE

F4/80

Page 15: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

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

Page 16: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MACROPHAGE INFILTRATION: FLOW CYTOMETRY

• SVC: Stromal Vascular cells• F4/80= macrophage marker• less macrophage infiltration in adipose tissue of anti-OPN group

Page 17: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MACROPHAGE INFILTRATION: TUNEL ASSAYTerminal deoxynucleotidyl

transferase-mediated d-UTP Nick End Labeling

Apoptosis “nicks” in DNA

Page 18: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

MACROPHAGE INFILTRATION: TUNEL ASSAY

Enhanced apoptosis in anti-OPN-treated mice disappearance of macrophages

Page 19: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

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)

Page 20: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

OVERVIEW OF STUDY

1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR

2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay

3. Adipose Tissue Inflammation Western Blot

Page 21: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT

Page 22: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

ADIPOSE TISSUE INFLAMMATION: WESTERN BLOT cJun NH2-terminal kinase (JNK) signaling

pathway required in macrophages to establish insulin resistance and inflammation

Phosphorylated JNK activated

Page 23: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

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

Page 24: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

SUMMARY OF FINDINGS

Antibody-mediated OPN neutralizationEnhanced insulin sensitivity Inhibited macrophage

accumulation in adipose tissue (apoptosis)

Attenuated adipose tissue inflammation

Page 25: N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,

BIG PICTURE

Inflammation insulin resistance metabolic diseases

OPN in obesityInflammation attract

macrophages into adipocytesOPN neutralization

stop influx of macrophages less inflammation better insulin sensitivity


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