Micro-RNA 132 and 212 mediated regulation of fatty acid metabolism and its effect on insulin secretion. Prelim Mock Talk by -Mufaddal S Soni Attie Lab

Outline Introduction Preliminary Data Specific Aims: Conclusion Diabetes Micro-RNAs Preliminary Data Specific Aims: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Conclusion The Economist, 12/13/03

Obesity-diabetes dichotomy Lean Obese B6 BTBR Obese Diabetic Age (weeks) 3

Micro-RNA 132 and 212 enhances insulin secretion.

Micro-RNA processing Transcription of pri-miRNA, Pri-miRNA processed by Drosha and DGCR8/Pasha, giving pre-miRNA, Pre-miRNA exported using exportin-5, Processed by dicer in the cytosol to give mature ds-miRNA. Ss-miRNA formed and incorporated into RISC. RISC degrades/translationally represses complementary RNA. Chang, Z. C. 2005

CACT (Slc25a20) : Most down regulated target of miRNA 132 and 212 Slc25a20 is the gene id for Cartnitine Acyl-Carnitine Translocase (CACT). Cells were profiled 10 and 24hrs after miRNA-132 and 212 overexpression. Red colored genes have seed region for the miRNAs.

CACT (Slc25a20) : Most down regulated target of miRNA 132 and 212 siCACT mimics the effect of miRNA on GSIS.

CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation.

CACT mediates translocation of FA-Carn into the mitochondria for β-oxidation.

Fatty acyl carnitine enhances insulin secretion 50µM 10 mM Long chain carnitines are more sensitive towards enhancing insulin secretion.

Fatty acyl carnitine enhances insulin secretion 50µM 50µM 10 mM

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Aim 1: Determine the molecular fate of LC-Carn when stimulating insulin secretion. Experiment 1: Decrease CPT-1 activity and determine if LC-Carn stimulates insulin secretion. Experiment 2: Determine if siRNA-mediated knockdown of CACT leads to reduced fatty acid β-oxidation. Experiment 3: Determine the molecular fate of exogenous FA-Carn under conditions when CACT activity is diminished. Experiment 4: Determine if a non-metabolized analog of fatty acyl-carnitine is capable of stimulating insulin secretion.

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion.

Non-specific PKC inhibitor. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn Ro 31-8220 Non-specific PKC inhibitor. LC-Carn

Conventional and novel PKC inhibitor. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn PMA Conventional and novel PKC inhibitor. LC-Carn

Conventional PKC inhibitor. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn UNC-01 Conventional PKC inhibitor. LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn siRNAs against PKC isozymes responsible for LC-Carn mediated insulin secretion. LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn LC-Carn

Measure increase in cAMP levels Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn Measure increase in cAMP levels LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn H89 PKA inhibitor. LC-Carn

Inhibitor of Rap-GTP, an Epac effector. Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn Rap-GAP Inhibitor of Rap-GTP, an Epac effector. LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. LC-Carn LC-Carn Wortmannin PI3K inhibitor LC-Carn

Aim 2: Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Experiment 1: Determine if LC-Carns enhance insulin secretion through PKC activation. Experiment 2: Study the role of cAMP and PKA in LC-Carn effects on insulin secretion. Experiment 3: Determine if the insulin signaling pathway is involved in LC-Carn mediated insulin secretion. Experiment 4: Identified proteins phosphorylated as a result of LC-Carn mediated PKC/PKA activation. Experiment 5: Determine the role of protein acylation in LC-Carn mediated insulin secretion. Phospho-proteomics if kinases are involved. What is Acylation? Acylation inhibition has been shown to blunt LC-CoA mediated insulin secretion. Inhibitors of Acylation: Cerulenin and 2-Br-palmitate.

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Specific Aims Determine the molecular fate of LC-Carn when stimulating insulin secretion. Identify the underlying signaling pathway for how LC-Carn enhances insulin secretion. Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion.

Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Prof. Randall Mynatt, LSU.

Glucose Tolerance Test (GTT) Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Experiment 1: Measure insulin secretion and glucose clearance of β-cell CrAT KO mice. Repeat GTT and also track insulin levels using C-peptide measurements. Perform ITT to measure insulin sensitivity of the CrAT KO mice Glucose Tolerance Test (GTT) Repeat the GSIS experiment on isolated islets from the CrAT KO mice with glucose and various other secretagogues. Prof. Randall Mynatt, LSU.

Insulin Tolerance Test (ITT) Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Experiment 1: Measure insulin secretion and glucose clearance of β-cell CrAT KO mice. Insulin Tolerance Test (ITT) Repeat GTT and also track insulin levels using C-peptide measurements. Perform ITT to measure insulin sensitivity of the CrAT KO mice Repeat the GSIS experiment on isolated islets from the CrAT KO mice with glucose and various other secretagogues

Glucose Stimulated Insulin Secretion (GSIS) Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Experiment 1: Measure insulin secretion and glucose clearance of β-cell CrAT KO mice. Repeat GTT and also track insulin levels using C-peptide measurements. Glucose Stimulated Insulin Secretion (GSIS) Perform ITT to measure insulin sensitivity of the CrAT KO mice Repeat the GSIS experiment on isolated islets from the CrAT KO mice with glucose and various other secretagogues. Prof. Randall Mynatt, LSU.

Amplification Pathway Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Experiment 2: Determine the phase of insulin secretion altered in CrAT mice. Amplification Pathway (2nd Phase) Triggering Pathway (1st Phase)

Experiment 3: Metabolic profiling of the βCrAT mouse islets. Aim 3: Elucidate the role of Carnitine Acetyl Transferase (CrAT) in regulating insulin secretion. Experiment 3: Metabolic profiling of the βCrAT mouse islets. Fat animals have elevated carnitine pools in the muscle. I will profile the carnitine metabolites in the βCrAT mouse islets to determine the correlation between insulin secretion and carnitine levels. Koves, R. T. et.al. 2007

Thank You Acknowledgements: Questions?

biotin–HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide