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An Antidiabetic Thiazolidinedione Is A High Affinity Ligand For Peroxisome Proliferator-Activated Receptor G (Pparg

This study demonstrates that thiazolidinediones (TZDs), antidiabetic drugs, exert their effects by binding to and activating peroxisome proliferator-activated receptor gamma (PPARγ). The researchers found that TZDs selectively activate PPARγ using reporter gene assays. Binding assays also showed that TZDs have high affinity for PPARγ. This was the first evidence that TZDs work by binding to and activating PPARγ, helping explain their antidiabetic mechanisms such as increasing glucose disposal and adipocyte differentiation.

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28 views

An Antidiabetic Thiazolidinedione Is A High Affinity Ligand For Peroxisome Proliferator-Activated Receptor G (Pparg

This study demonstrates that thiazolidinediones (TZDs), antidiabetic drugs, exert their effects by binding to and activating peroxisome proliferator-activated receptor gamma (PPARγ). The researchers found that TZDs selectively activate PPARγ using reporter gene assays. Binding assays also showed that TZDs have high affinity for PPARγ. This was the first evidence that TZDs work by binding to and activating PPARγ, helping explain their antidiabetic mechanisms such as increasing glucose disposal and adipocyte differentiation.

Uploaded by

shamshir007
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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An antidiabetic thiazolidinedione is a high affinity

ligand for peroxisome proliferator-activated


receptor g (PPARg)

Jurgen Lehmann et al., 1995, Journal of Biological Chemistry


PPAR
• Ligand-activated nuclear transcription
factors
• 3 subtypes
– PPAR a -liver and skeletal muscle
– PPAR g -adipose tissue
– PPAR d – most cell types (aka NUC-1)
• Regulate transcription of genes involved in
fatty acid oxidation and storage
membrane

PPAR RXR

cytosol

nucleus
Increased transcription
of b-ox genes
DNA
PPAR Response element
TZDs
Antidiabetic agents
oral hypoglycemic
reduce insulin resistance
increase glucose uptake
decrease hepatic glucose output
induce adipocyte differentiation
- mature adipocyte
Signficance
• This is the first paper to demonstrate that
TZDs exert their effects by binding and
activating PPAR g

• Experiment 1:Determine if TZDs can


selectively activate PPARg
Methods: cDNA constructs
PPAR cDNA placed in expression vector (pSG5)

CAT reporter to monitor PPAR activity


Contains GAL4 DNA binding element

B-galactosidase reporter?

CV-1 cells Kidney cells from male adult African green monkey
Does BRL49653 bind to PPARg?
GST-PPAR (LBD) cDNA

ALLOWS
FOR PURIFICATION
BINDS GLUTATHIONE

Grow in E.Coli

Prepare extracts/purify

Add radiolabel (3H BRL)

Remove unbound 3H BRL and count radioactivity


Binding Curves
• Affinity of a ligand for a receptor
• Measure the concentration of bound ligand
to a known amount of binder
– Ligand is BRL49653 (TZD)
– Binder is PPARg
Figure 3 A

Total binding
Specific
binding

Excess
BRL49653
Scatchard Plot
• method for analyzing data for freely
reversible ligand/receptor binding
interactions.
• Make data linear – nice to look at
Kd – indicates the strength of binding between PPAR and BRL
small Kd indicates a very strong interaction
Competitive Binding Assays

Ligand (3H BRL) Ligand (3H BRL)

receptor receptor
Add known amount of
labeled BRL49653

Compete off with various


compounds
Conclusion
• TZD targets PPARg
• Diverse functions
– Glucose utilization
– Adipocyte differentiation
• “Increases in lipid levels have been shown
to interfere with glucose disposal”
– Ectopic fat theory

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