1,25-‐Dihydroxyvitamin D3 and Re7noic Acid Modula7on of PPARγ Expression and Insulin
Resistance in Diabetes Mellitus II
Brian R. Covello
Diabetes Mellitus II • 25.8 million people • By 2050, 1 in 3 US adults will have diabetes (Powers,
2005)
• Leading cause of kidney failure, blindness, non-‐accidental amputa7ons of legs (Powers, 2005)
• Insulin – Beta cells, islet of Langerhans in Pancreas – Regulates glucose metabolism
• Uptake of glucose • Decrease in gluconeogenesis • Insulin Resistance à DM II (Powers, 2005)
• Links to obesity (Wajchemberg, 2000)
• TZDs are efficient an7-‐diabe7c drugs
PPARγ • Nuclear Receptor • Transcrip7on factors for DNA
• Heterodimerize with RXR • Increase insulin sensi7vity (Liang, 2006)
• Increase adipocyte differen7a7on (Liang, 2006)
• Increase fat uptake • PPARγ1 and PPARγ2 (isoforms) • Promoter usage and splicing (Liang, 2006)
• Co-‐ac7vators, co-‐repressors, ligands, phosphoryla7on (Liang, 2006)
Vitamin D • Vitamin D is a fat soluble vitamin
• Vitamin D insufficiency correlated to obesity and DM II
• UV à epithelial cells à Vitamin D à 1,25-‐dihydroxyvitamin D3 à VDR
(Yoshifumi, 1998)
Vitamin A
• Re7nol A is fat soluble vitamin A • Fat soluble? • 3T3-‐L1
• Once inside cells metabolized to Re7noic Acid • Inhibitory Effect on PPAR gamma
(Yoshifumi, 1998)
No7ce Anything?
ß PPAR gamma
Re7noic Acid 1,25-‐dihydroxyvitamin D3
• Limited supply of RXR receptor • Compe77on amongst nuclear receptors for a common heterodimeric partner (Yoshifumi, 1998)
Previous Research
• Muta7ons in PPAR gamma cause insulin resistance (Barroso, 1999)
• TZDs bind PPAR gamma (Gregoire, 1998) • 1,25(OH)2D3 has inhibitory effect on differen7a7on and prolifera7on in 3T3-‐L1 cells (Yoshifumi, 1998)
– Inhibi7on of PPAR gamma expression • Re7noic acid also slightly inhibits PPAR (Yoshifumi, 1998) • Tests were conducted on cells proceeding through differen7a7on process
• Tests were under the influence of PPAR ligand TZD
Previous Research
• In 1988 Ishida et al reported inhibitory effect on prolifera7on and differen7a7on of 3T3-‐L1 cells – 67% decrease at 10-‐8M 1,25(OH)2D3
– Significant decrease even at 10-‐10M – Exact mechanism was s7ll unknown
• 1998 Yoshfumi et al showed 1,25(OH)2D3 inhibited PPAR gamma expression when bound to TZD – Vitamin D insufficiency à More PPAR à More Adipocyte Differen7a7on à Obesity à DM II
Gaps & Goals • No test of mixtures of metabolites has been conducted – So what? – Mixtures are found in vivo
• No tests were conducted on 3T3-‐L1 cells during pre-‐adipocyte stage
• No tests on non-‐ligand bound PPAR have been conducted
• No transac7va7on studies have been conducted
Stage 1 • Test 1,25(OH)2D3 and Re7noic Acid combina7ons in pre-‐adipocytes
Treat 3T3-‐L1 cells Control: No tx Dish 1: 1x10-‐9 M 1,25-‐dihydroxyvitamin D3 Dish 2: 1x10-‐9 M re7noic acid Dish 3: 1x10-‐6 M 1,25-‐dihydroxyvitamin D3 Dish 4: 1x10-‐6 M re7noic acid Dish 5: 1x10-‐9 M each of 1,25-‐dihydroxvitamin D and re7noic acid Dish 6: 1x10-‐6 M each of 1,25-‐dihydroxvitamin D and re7noic acid Dish 7: 1x10-‐9 M of 1,25-‐dihydroxvitamin D3 and 1x10-‐6 M re7noic acid Dish 8: 1x10-‐6 M of 1,25-‐dihydroxyvitamin D3 and 1x10-‐9 M re7noic acid Times: 0hours (control), 8 hours, 20 hours, 40 hours
Hypothesis: Mixed concentra7ons = Greater Inhibi7on & Less D3 needed for inhibi7on than previously suggested
Stage 2
• Induce differen7a7on of 3T3-‐L1 cells into adipocytes
• Test mixture of metabolites on induced cells • Conduct GDPH Assay – Glycerol 3-‐Phosphate Dehydrogenase
• Direct link to obesity and diabetes
Stage 3 • Transac7va7on studies • Up-‐regula7on – adipocyte faly acid-‐binding protein
– acyl-‐CoA synthase – lipoprotein lipase – c-‐Cbl associa7ng protein – phosphoenolpyruvate carboxykinase
– faly acid transport protein – insulin receptor substrate 2
(Wajchenberg, 2000)
Methods • Western Blot
– Protein Lysate – Enumera7on through spectrophotometry
– SDS-‐PAGE à PVDF Membrane
– Polyclonal Ab and Secondary Ab w/ BCIP
– Counterstain Ac7n • Immunofluorescence
– Characterize structural changes (nuclear receptor)
(Bogazzi, 2007)
(Yoshifumi, 1998)
Sources Barroso, I. B. (1999). Dominant nega7ve muta7ons in human ppar gamma associated with sever insulin resistance, diabetes
mellitus and hypertension. Le(ers to Nature, 402(23), 880-‐889. Liang, G. L. (2006). Peroxisome proliferator ac7vated receptor gamma as a drug target in the pathogenesis of insulin resistance.
Pharmacology & Therapeu<cs, 111(4), 145-‐173. Ishida, Y. (1988). Possible involvement of 1,25-‐dihydroxyvitamine d3 in prolifera7on and differen7a7on of 3t3-‐l1 cells.
Biochemical and Biophysical Research Communica<ons, 151(3), 1122-‐1127. Yoshifumi. (1998). Counterac7on of re7noic acid and 1,25-‐dihydroxyvitamin d3 on up-‐regula7on of adipocyte differen7a7on with
ppar ligand, an an7diabe7c thiazolidinedione, in 3t3-‐l1 cells. Pharmacology Le(ers, 62(14), 205-‐211. Powers, A. C. (2005). Chapter 323. Diabetes mellitus. In D. L. Kasper, A. S. Fauci, D. L. Longo, E. Braunwald, S. L. Hauser, & J. L.
Jameson, Harrison’s principles of internal medicine (16th ed.). The McGraw-‐Hill Companies, Inc. Wajchenberg, B. L. (2000). Subcutaneous and visceral adipose 7ssue: their rela7on to the metabolic syndrome. Endocr Rev 21,
697–738. Bogazzi, F. (2007). Abnormal expression of ppar gamma isoforms in the subcutaneous adipose 7ssue of pa7ents with cushing's
disease. Clinical Endocrinology, 1365(66), 7-‐12.