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

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Presentation on theme: "N EUTRALIZATION OF O STEOPONTIN I NHIBITS O BESITY -I NDUCED I NFLAMMATION AND I NSULIN RESISTANCE Florian W. Kiefer, Maximilian Zeyda, Karina Gollinger,"— Presentation transcript:

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, Birgit Pfau, Angelika Neuhofer, Thomas Weichhart, Marcus D. Saemann, Rene Geyeregger,1 Michaela Schlederer,3 Lukas Kenner, and Thomas M. Stulnig

2 W HAT IS O STEOPONTIN (OPN)? Glycosylated phosphoprotein ( SPP1 gene) Pro-inflammatory cytokine Functions: Bone formation Inflammation Cell migration Implication in diseases

3 O BESITY & I NFLAMMATION Chronic low-grade inflammation Inflammation  insulin resistance  metabolic diseases

4 O BESITY, I NFLAMMATION, O STEOPONTIN OPN

5 O BJECTIVE OF S TUDY OPN neutralizing antibody Hypothesis Targeting OPN in obesity could reverse adipose tissue inflammation and insulin resistance

6 M ICE M ODELS ( C57BL/6) Induced Obesity  24 weeks High Fat Diet (HFD) Normal Chow (NC) HFD NC Control Antibody OPN- neutralizing Antibody OPN- neutralizing Antibody Control Antibody

7 N EUTRALIZING A NTIBODY Goat Anti-Mouse OPN-specific antibodies Control Goat antibody MOUSE OPN Production of antibodies against mouse OPN

8 O VERVIEW OF S TUDY 1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR 2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay 3. Adipose Tissue Inflammation Western Blot

9 I NSULIN S ENSITIVITY : I NSULIN T OLERANCE T EST Assess response to insulin Fast 3 hrs  blood glucose levels measured

10 I NSULIN S ENSITIVITY : HOMA-IR H omeostasis M odel A ssessment of I nsulin R esistance HOMA-IR values  insulin sensitivity high “ “  insulin resistance

11 I NSULIN S ENSITIVITY OPN neutralization  enhanced insulin sensitivity ITT Anti-OPN group HF  respond better to insulin HOMA-IR IR significantly lower after anti- OPN treatment

12 O VERVIEW OF S TUDY 1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR 2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay 3. Adipose Tissue Inflammation Western Blot

13 M ACROPHAGE I NFILTRATION : RT-PCR S YBR G REEN

14 M ACROPHAGE I NFILTRATION : I MMUNOFLUORESCENCE F4/8 0

15 M ACROPHAGE I NFILTRATION : F LOW C YTOMETRY Peripheral Blood Addition of antibody Skin Alignment of cells in single file Cell Culture Fluid Portion Charge Assigner Adipose Stromal Vascular Fraction Stromal Vascular Fraction

16 M ACROPHAGE I NFILTRATION : F LOW C YTOMETRY SVC : Stromal Vascular cells F4/80 = macrophage marker less macrophage infiltration in adipose tissue of anti-OPN group

17 M ACROPHAGE I NFILTRATION : TUNEL A SSAY T erminal deoxynucleotidyl transferase- mediated d- U TP N ick E nd L abeling Apoptosis  “nicks” in DNA

18 M ACROPHAGE I NFILTRATION : TUNEL A SSAY Enhanced apoptosis in anti-OPN- treated mice  disappearance of macrophages

19 M ACROPHAGE ACCUMULATION IN A DIPOSE T ISSUE OPN Neutralization  inhibits macrophage infiltration RT-PCR macrophage marker mRNA Immunofluorescence lit areas on adipose tissue Flow Cytometry % F4/80 + cells of SVC TUNEL apoptosis (HF anti-OPN)

20 O VERVIEW OF S TUDY 1. Insulin Sensitivity Insulin Tolerance Test HOMA-IR 2. Macrophage Infiltration RT-PCR Immunofluorescence Flow Cytometry TUNEL Assay 3. Adipose Tissue Inflammation Western Blot

21 A DIPOSE T ISSUE I NFLAMMATION : W ESTERN B LOT

22 cJun NH2-terminal kinase (JNK) signaling pathway  required in macrophages to establish insulin resistance and inflammation Phosphorylated JNK  activated

23 A DIPOSE T ISSUE I NFLAMMATION : 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

24 SUMMARY OF FINDINGS Antibody-mediated OPN neutralization Enhanced insulin sensitivity Inhibited macrophage accumulation in adipose tissue (apoptosis) Attenuated adipose tissue inflammation

25 BIG PICTURE Inflammation  insulin resistance  metabolic diseases OPN in obesity Inflammation  attract macrophages into adipocytes OPN neutralization stop influx of macrophages  less inflammation  better insulin sensitivity


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