An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor g (PPARg)

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An antidiabetic thiazolidinedione is a high
affinity ligand for peroxisome proliferator-activa
ted receptor g (PPARg)
Jurgen Lehmann et al., 1995, Journal of
Biological Chemistry
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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

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membrane
RXR
PPAR
cytosol
nucleus
Increased transcription of b-ox genes
DNA
PPAR Response element
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TZDs

• Antidiabetic agents
• oral hypoglycemic
• reduce insulin resistance
• increase glucose uptake
• decrease hepatic glucose output
• induce adipocyte differentiation
• - mature adipocyte

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Signficance

• This is the first paper to demonstrate that TZDs
  exert their effects by binding and activating
  PPAR g
• Experiment 1Determine if TZDs can selectively
  activate PPARg

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Methods cDNA constructs
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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
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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
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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

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Figure 3 A
Total binding
Specific binding
Excess BRL49653
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Scatchard Plot

• method for analyzing data for freely reversible
  ligand/receptor binding interactions.
• Make data linear nice to look at

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Kd indicates the strength of binding between
PPAR and BRL small Kd indicates a very
strong interaction
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Competitive Binding Assays
Ligand (3H BRL)
Ligand (3H BRL)
receptor
receptor
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Add known amount of labeled BRL49653 Compete off
with various compounds
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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