brian covello: diabetes research introduction
DESCRIPTION
Brian Covello's preliminary presentation on DMII research. Diabetes mellitus (DM) also known as simply diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period.[2] This high blood sugar produces the symptoms of frequent urination, increased thirst, and increased hunger. Untreated, diabetes can cause many complications.[3] Acute complications include diabetic ketoacidosis and nonketotic hyperosmolar coma.[4] Serious long-term complications include heart disease, stroke, kidney failure, foot ulcers and damage to the eyes.[3] Diabetes is due to either the pancreas not producing enough insulin, or the cells of the body not responding properly to the insulin produced.[5] There are three main types of diabetes mellitus: Type 1 DM results from the body's failure to produce enough insulin. This form was previously referred to as "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes". The cause is unknown.[3] Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly.[3] As the disease progresses a lack of insulin may also develop.[6] This form was previously referred to as non insulin-dependent diabetes mellitus (NIDDM) or "adult-onset diabetes". The primary cause is excessive body weight and not enough exercise.[3] Gestational diabetes, is the third main form and occurs when pregnant women without a previous history of diabetes develop a high blood glucose level.[3] Prevention and treatment involves a healthy diet, physical exercise, not using tobacco, and being a normal body weight. Blood pressure control and proper foot care are also important for people with the disease. Type 1 diabetes must be managed with insulin injections.[3] Type 2 diabetes may be treated with medications with or without insulin.[7] Insulin and some oral medications can cause low blood sugar.[8] Weight loss surgery in those with obesity is an effective measure in those with type 2 DM.[9] Gestational diabetes usually resolves after the birth of the baby.[10] Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases.[11][12] This is equal to 8.3% of the adults population,[12] with equal rates in both women and men.[13] Worldwide in 2012 and 2013 diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death.[7][14] Diabetes overall at least doubles the risk of death.[3] The number of people with diabetes is expected to rise to 592 million by 2035.[15] The economic costs of diabetes globally was estimated in 2013 at $548 billion[14] and in the United States in 2012 $245 billion The classic symptoms of untreated diabetes are weight loss, polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger).[17]TRANSCRIPT
1,25-‐Dihydroxyvitamin D3 and Re7noic Acid in vitro Modula7on of PPARγ and Insulin Resistance in
Diabetes Mellitus II
Brian Covello
Mo7va7on • 25.8 million people affected by DM • By 2050, 1 in 3 US adults will be affected • Normal human physiology gone wrong – Glucose à Insulin
• Beta cells, islet of Langerhans – Type I and Type II
• Linked to Obesity • TZDs as an7-‐diabe7c drugs
PPARγ
• RXR green • PPARγ purple
• 57.6kDa • Adipogenesis, Insulin
sensi7vty
1,25-‐Dihydroxyvitamin D3 and Re7noic Acid in vitro Modula7on of PPARγ and Insulin Resistance in
Diabetes Mellitus II
Brian Covello
• Limited supply of RXR • VDR and RAR heterodimerize with RXR
• Fierce compe77on! • Ligands – VDR = D3 – RAR = RA
The Connec7on
What Do We Know?
• Vitamin D down regulates expression of PPARγ (Yoshifumi, 1998) – Vitamin D correlated to Obesity
• Re7noic Acid also inhibits PPARγ (Yoshifumi, 1998)
• RA and D3 effects tested separately • Muta7ons in PPARγ cause insulin resistance (Barroso, 1999)
The Research • Diabetes Mellitus Type II • In vitro analysis, in vivo predic7ons • A bridge to transla7onal research • Mimic human physiology • D3 and RA tested together – first 7me! • Nanomolar human serum concentra7on
• Hypothesis: Synergy of down regula7on of PPARγ when D3 and RA are combined
• Implica7ons: Diet & Drug interac7on
Bidirec7onal Approach
• Immunofluorescence Microscopy – Delocaliza7on of PPARγ – IF never conducted in this manner – Unable to indicate up/down regula7on
• SDS-‐PAGE – Separa7on of proteins based on size – Protein changes? – Up/down regula7on à Western Blot
Immunofluorescence Microscopy
• Fixed à Permeabilized à Blocked • Primary AB = rabbit an7-‐PPAR
– BD transduc7on labs – 1:25 concentra7on
• Secondary AB = goat an7-‐rabbit SFX kit – Alexa Fluora, 1:25
• + control 1% EtOH à delocaliza7on (Powers, 2005)
• -‐ Control Incomplete
Tx 1: Incomplete Tx 2: 1% EtOH Tx 3: 1 μM D3 Tx 4: 1 μM RA Tx 5: 1 nm D3 Tx 6: 1 nm RA Tx 7: 1 μM D3/RA Tx 8: 1 nM D3/RA
I Swear… I didn’t kill them
• Proof: • Elongated • Nuclei present and dis7nct • Amount of cells per frame the same
• 40X • Canon • B. Covello
SDS-‐PAGE
• Gives false nega7ve charge to proteins • Separated based on size • Proteins migrate toward + end • 50mA • Make sure to put same protein amount in each well…
Standard Curve
• 50 micrograms
• Disappearance of protein in combined • Novel protein changes in nanomolar
treatment that aren’t present in micromolar • Human serum levels
Where Do We Go From Here?
• Western Blot – What are those proteins? – PPARγ changes?
• 3T3-‐L1 – Pre-‐adipocytes – Closer to human physiology – Berer rela7on to DMII – Differen7a7on process
• Insulin, Dexamethosone, 3-‐isobutyl-‐1-‐methylxanthine (IBMX)
Acknowledgements
• FSC for funding • Dr. Bradshaw • Classmates
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.