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Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin resistance

Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin resistance.

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Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin resistance

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  1. 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, ReneGeyeregger,1 Michaela Schlederer,3Lukas Kenner,and Thomas M. Stulnig

  2. What is Osteopontin (OPN)? • Glycosylatedphosphoprotein (SPP1 gene) • Pro-inflammatory cytokine • Functions: • Bone formation • Inflammation • Cell migration • Implication in diseases

  3. Obesity & Inflammation • Chronic low-grade inflammation • Inflammation insulin resistance  metabolic diseases

  4. Obesity, Inflammation, Osteopontin OPN

  5. Objective of Study • OPN neutralizing antibody • Hypothesis • Targeting OPN in obesity could reverse adipose tissue inflammation and insulin resistance

  6. Mice Models (C57BL/6) • Induced Obesity 24 weeks High Fat Diet (HFD) • Normal Chow (NC) NC HFD OPN-neutralizing Antibody OPN-neutralizing Antibody Control Antibody Control Antibody

  7. Neutralizing Antibody • Goat Anti-Mouse OPN-specific antibodies • Control Goat antibody MOUSE OPN Production of antibodies against mouse OPN

  8. Overview of Study • Insulin Sensitivity • Insulin Tolerance Test • HOMA-IR • Macrophage Infiltration • RT-PCR • Immunofluorescence • Flow Cytometry • TUNEL Assay • Adipose Tissue Inflammation • Western Blot

  9. Insulin Sensitivity: Insulin Tolerance Test • Assess response to insulin • Fast 3 hrs blood glucose levels measured

  10. Insulin Sensitivity: HOMA-IR • Homeostasis Model Assessment of Insulin Resistance • HOMA-IR values insulin sensitivity high • “ “  insulin resistance

  11. Insulin Sensitivity • OPN neutralization enhanced insulin sensitivity • ITT • Anti-OPN group HF respond better to insulin • HOMA-IR • IR significantly lower after anti-OPN treatment

  12. Overview of Study • Insulin Sensitivity • Insulin Tolerance Test • HOMA-IR • Macrophage Infiltration • RT-PCR • Immunofluorescence • Flow Cytometry • TUNEL Assay • Adipose Tissue Inflammation • Western Blot

  13. Macrophage Infiltration: RT-PCR Sybr Green

  14. Macrophage Infiltration: Immunofluorescence F4/80

  15. Macrophage Infiltration: Flow Cytometry Adipose Adipose Addition of antibody Peripheral Blood Fluid Portion Skin Stromal Vascular Fraction Alignment of cells in single file Cell Culture Charge Assigner

  16. Macrophage Infiltration: Flow Cytometry • SVC: Stromal Vascular cells • F4/80= macrophage marker • less macrophage infiltration in adipose tissue of anti-OPN group

  17. Macrophage Infiltration: TUNEL Assay • Terminal deoxynucleotidyltransferase-mediated d-UTP Nick End Labeling • Apoptosis “nicks” in DNA

  18. Macrophage Infiltration: TUNEL Assay • Enhanced apoptosis in anti-OPN-treated mice  disappearance of macrophages

  19. Macrophage accumulation in Adipose Tissue • 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. Overview of Study • Insulin Sensitivity • Insulin Tolerance Test • HOMA-IR • Macrophage Infiltration • RT-PCR • Immunofluorescence • Flow Cytometry • TUNEL Assay • Adipose Tissue Inflammation • Western Blot

  21. Adipose Tissue Inflammation: Western Blot

  22. Adipose Tissue Inflammation: Western Blot • cJun NH2-terminal kinase (JNK) signaling pathway required in macrophages to establish insulin resistance and inflammation • Phosphorylated JNK activated

  23. 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

  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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