Neutralization of osteopontin inhibits obesity induced inflammation and insulin resistance
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Neutralization of osteopontin inhibits obesity induced inflammation and insulin resistance

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


What is osteopontin opn
What is Osteopontin (OPN)? Inflammation and Insulin resistance

  • Glycosylatedphosphoprotein (SPP1 gene)

  • Pro-inflammatory cytokine

  • Functions:

    • Bone formation

    • Inflammation

    • Cell migration

  • Implication in diseases


Obesity inflammation
Obesity & Inflammation Inflammation and Insulin resistance

  • Chronic low-grade inflammation

  • Inflammation insulin resistance  metabolic diseases


Obesity inflammation osteopontin
Obesity, Inflammation, Osteopontin Inflammation and Insulin resistance

OPN


Objective of study
Objective of Study Inflammation and Insulin resistance

  • OPN neutralizing antibody

  • Hypothesis

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


Mice models c57bl 6
Mice Models ( Inflammation and Insulin resistanceC57BL/6)

  • Induced Obesity 24 weeks High Fat Diet (HFD)

  • Normal Chow (NC)

NC

HFD

OPN-neutralizing

Antibody

OPN-neutralizing

Antibody

Control

Antibody

Control

Antibody


Neutralizing antibody
Neutralizing Antibody Inflammation and Insulin resistance

  • Goat Anti-Mouse OPN-specific antibodies

  • Control Goat antibody

MOUSE OPN

Production of antibodies against mouse OPN


Overview of study
Overview of Study Inflammation and Insulin resistance

  • Insulin Sensitivity

    • Insulin Tolerance Test

    • HOMA-IR

  • Macrophage Infiltration

    • RT-PCR

    • Immunofluorescence

    • Flow Cytometry

    • TUNEL Assay

  • Adipose Tissue Inflammation

    • Western Blot


Insulin sensitivity insulin tolerance test
Insulin Sensitivity: Inflammation and Insulin resistanceInsulin Tolerance Test

  • Assess response to insulin

  • Fast 3 hrs blood glucose levels measured


Insulin sensitivity homa ir
Insulin Sensitivity: Inflammation and Insulin resistance HOMA-IR

  • Homeostasis Model Assessment of Insulin Resistance

    • HOMA-IR values insulin sensitivity high

    • “ “  insulin resistance


Insulin sensitivity
Insulin Sensitivity Inflammation and Insulin resistance

  • OPN neutralization enhanced insulin sensitivity

    • ITT

      • Anti-OPN group HF respond better to insulin

    • HOMA-IR

      • IR significantly lower after anti-OPN treatment


Overview of study1
Overview of Study Inflammation and Insulin resistance

  • Insulin Sensitivity

    • Insulin Tolerance Test

    • HOMA-IR

  • Macrophage Infiltration

    • RT-PCR

    • Immunofluorescence

    • Flow Cytometry

    • TUNEL Assay

  • Adipose Tissue Inflammation

    • Western Blot


Macrophage infiltration rt pcr sybr green
Macrophage Infiltration: Inflammation and Insulin resistanceRT-PCR Sybr Green


Macrophage infiltration immunofluorescence
Macrophage Infiltration: Inflammation and Insulin resistanceImmunofluorescence

F4/80


Macrophage infiltration flow cytometry
Macrophage Infiltration: Inflammation and Insulin resistanceFlow Cytometry

Adipose

Adipose

Addition of antibody

Peripheral Blood

Fluid Portion

Skin

Stromal Vascular

Fraction

Alignment of cells in single file

Cell Culture

Charge Assigner


Macrophage infiltration flow cytometry1
Macrophage Infiltration: Inflammation and Insulin resistanceFlow Cytometry

  • SVC: Stromal Vascular cells

  • F4/80= macrophage marker

  • less macrophage infiltration in adipose tissue of anti-OPN group


Macrophage infiltration tunel assay
Macrophage Infiltration: Inflammation and Insulin resistanceTUNEL Assay

  • Terminal deoxynucleotidyltransferase-mediated d-UTP Nick End Labeling

  • Apoptosis “nicks” in DNA


Macrophage infiltration tunel assay1
Macrophage Infiltration: Inflammation and Insulin resistanceTUNEL Assay

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


Macrophage accumulation in adipose tissue
Macrophage accumulation in Adipose Tissue Inflammation and Insulin resistance

  • 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)


Overview of study2
Overview of Study Inflammation and Insulin resistance

  • Insulin Sensitivity

    • Insulin Tolerance Test

    • HOMA-IR

  • Macrophage Infiltration

    • RT-PCR

    • Immunofluorescence

    • Flow Cytometry

    • TUNEL Assay

  • Adipose Tissue Inflammation

    • Western Blot


Adipose tissue inflammation western blot
Adipose Tissue Inflammation: Inflammation and Insulin resistanceWestern Blot


Adipose tissue inflammation western blot1
Adipose Tissue Inflammation: Inflammation and Insulin resistanceWestern Blot

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

  • Phosphorylated JNK activated


Adipose tissue inflammation
Adipose Tissue Inflammation: Inflammation and Insulin resistance

  • 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 findings
SUMMARY OF FINDINGS Inflammation and Insulin resistance

  • Antibody-mediated OPN neutralization

    • Enhanced insulin sensitivity

    • Inhibited macrophage accumulation in adipose tissue (apoptosis)

    • Attenuated adipose tissue inflammation


Big picture
BIG PICTURE Inflammation and Insulin resistance

  • Inflammation  insulin resistance metabolic diseases

  • OPN in obesity

    • Inflammation attract macrophages into adipocytes

  • OPN neutralization

    • stop influx of macrophages less inflammation  better insulin sensitivity