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Transcript
Lipodystrophy: Metabolic and Clinical Aspects Resource Room Slide Series Cellular Pathology of Insulin Resistance in Lipodystrophy Robert R. Henry, MD Professor of Medicine University of California, San Diego VA San Diego Healthcare System San Diego, CA Theodore P. Ciaraldi, PhD Project Scientist, UCSD La Jolla, CA Disclosures • Dr. Henry: – Grants: AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Medtronic, and Sanofi – Consulting: Boehringer Ingelheim, Eli Lilly, Gilead, Intarcia, Isis, Novo Nordisk, Roche/Genentech, and Sanofi – Advisory board: Amgen, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Elcelyx, Eli Lilly, Gilead, Intarcia, Johnson & Johnson/Janssen, Merck, Novo Nordisk, Roche/Genentech, Sanofi • Dr. Ciaraldi – There are no relevant financial relationships to disclose. Objectives • Understand the role of ectopic fat storage in the development of insulin resistance • Understand the nature of bi-directional signaling that occurs between adipose tissue and other organs: muscle, liver, and pancreas • Recognize how post-translational modifications (PTMs) affect both signaling pathways and gene regulation Overview 1. Fat storage • Where and how? • Fat metabolism in skeletal muscle • Disordered fat metabolism 2. Insulin signaling • • • • Target tissues and responses Pathways PTMs Disordered insulin signaling 3. Adipose tissue-skeletal muscle communication • Activation of stress kinases • Altered phosphorylation of insulin receptor substrate 1 (IRS-1): consequences • Altered PTMs of transcription factors: consequences Body Fat Distribution Healthy Subcutaneous obesity Visceral obesity Lipodystrophy Definitions Ectopic: Ectopic Fat: Occurring in an abnormal position or unusual manner or form* Abnormal fat accumulation other than in adipose tissue (eg, skeletal muscle, heart, liver, pancreas) * Merriam-Webster dictionary Fat Distribution Between Tissues Adipose Tissue Compartment Plasma Muscle Compartment Lean Subcutaneous Obesity Visceral Obesity Lipodystrophy Unger RH. Trends Endocrinol Metab. 2003;14:398-403. Intramyocellular Lipid Relationship to Insulin Sensitivity ● Insulin Sensitivity (clamp log10 mol/l) (mg/min kg FFM + 17.7) 0.9 r = -0.53 P<0.0006 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Current Opinion in Pharmacology 1 2 3 4 5 6 7 8 9 10 Skeletal Muscle-Associated Triglyceride (mmol/g wet weight of tissue) Nawrocki AR, Scherer PE. Curr Opin Pharmacol. 2004;4:281-289. Kelley DE, Goodpaster BH. Diabetes Care. 2001;24:933-941. Lipid Droplets (LDs) Multi-Faceted Organelles for Lipid Storage and Mobilization • Outer phospholipid monolayer • Neutral lipid core: triglycerides, cholesterol esters • Monolayer studded with LD proteins: – Lipases and activators – Adipocyte triglyceride lipase (ATGL) – Hormone sensitive lipase (HSL) – Comparative gene identification-58 (CGI58), activator of ATGL – Perilipins (PLINs) 1-5: regulate access of lipases to lipid tissue-specific expression – LDs permit organization and regulation of lipid storage and mobilization Lipid Storage in Cells Adipocyte Skeletal muscle LD LD LD - HSL - ATGL - CGI58 - PLIN 1 - PLIN 2 LD - PLIN 5 Sources and Fates of Lipids and Fats Tissue Specificity Tissue Lipid Source Fate Adipose tissue Internal stores Circulation Synthesis Lipolysis and release to circulation Structural Skeletal muscle Circulation Internal stores (Limited) Oxidation Structural Liver Circulation Internal stores (Limited) Synthesis Oxidation Lipoprotein production Structural Pancreas Circulation Oxidation Fat Metabolism in Muscle Healthy Hyperlipidemia FFA FFA FATP1 FAT FABPpm FATP1 CO2 FABPpm CO2 Ceramide FFA Ceramide FFA mito LCFACoA FAT LD Diffusion DAG DAG LCFACoA Protein-mediated transport and trafficking Fats and Metabolites in Muscle Change in Lipodystrophy Intramyocellular lipid Triglyceride (TG) Diacylglycerol (DAG) * Long Chain Fatty Acyl CoA (LCFA-CoA) Ceramide Acylcarnatines CO2 * Degree of fatty acid (FA) saturation altered in insulin-resistant conditions Fat Cell Functions Then Lipoprotein LPL FFA FFA FFA (in lipodystrophy) TG Glucose LD Adipose Tissue Functions Now Angiotensin II Visfatin CRP PAI-1 Increased in lipodystrophy FAT FFA IL-6 MCP-1 TNF-α Decreased in Lipodystrophy Adiponectin Leptin Resistin Summary, Part 1 • In healthy individuals, lipid is stored predominately in adipose tissue, with subcutaneous adipose tissue > visceral adipose tissue. • Lipid storage in muscle, liver, and pancreas is limited and present primarily to meet energetic and structural needs. • Lipid storage in tissues is tightly regulated, primarily through the actions of proteins associated with LDs. Summary, Part 1, cont. • Loss of the storage capacity of adipose tissue, such as in lipodystrophy, leads to increased lipid stores in other tissues (ectopic fat). • Lipid oversupply results in altered/incomplete FFA oxidation. • Specific FA metabolites can have deleterious effects on cellular function, including secretion (eg, adipokines). Insulin Target Tissues – 1 Tissue Skeletal muscle Adipose tissue Response Glucose uptake Glycogen synthesis Lipolysis Lipogenesis Glucose production Liver Lipogenesis Lipoprotein production Glucose oxidation Heart FFA oxidation Hypertrophy Insulin Target Tissues – 2 Tissue Brain Response Appetite Sympathetic tone Vasoconstriction Endothelium Vasodilation Leukocyte adhesion Macrophage Low density lipoprotein uptake Insulin Signaling Cast of Players IR Insulin Receptor GLUT4 Glucose Transporter 4 IRS (1-5) Insulin Receptor Substrate GSK3 Glycogen Synthase Kinase 3 PI3-K Phosphoinositide 3 Kinase (p85 & p110 kDa Subunits) Grb2 Growth Factor Receptor Bound-2, Scaffold Protein PDK1 Phosphoinositide-Dependent Kinase SOS Son of Sevenless, Scaffold Protein Also known as Protein Kinase B ERK Extracellular Regulated Kinase Akt aPKC Atyptical Protein Kinase C mTOR Mammalian Target of Rapamycin AS160 Akt Substrate of 160 kDa Shc Src homology 2 and collagen-like Insulin Receptor -S-S- subunit Plasma Membrane Insulin Binding - Transmembrane Region ß subunit Y953 Y960 - Juxtamembrane Region Y1146 Y1150 Y1151 - Kinase Regulatory Region Y1316 Y1322 - C-Terminal Regulatory Region Adapted from Ciaraldi TP. In Principles of Diabetes Mellitus. 2nd ed. Poretsky LO, ed. New York, Springer,2009, p. 75-87. Principles of Insulin Signaling • The IR can phosphorylate both itself and other proteins (eg, IRS) on the amino acid tyrosine. • Phosphorylation creates recognition sites for IRS and other proteins to serve as scaffolds, recruiting other molecules into signaling complexes. • Phosphorylation on other sites can oppose complex formation. Principles of Insulin Signaling, cont. • Intracellular localization of these multi-molecular signaling complexes plays an important role in the subsequent pathways activated. • Phosphorylation is just one of a number of posttranslational modifications (PTMs) that can influence complex formation, sub-cellular localization, enzyme activity, and stability or degradation of the protein(s). PTMs of Proteins Modification Enzyme Responsible Kinase (eg, IR) P Phosphorylation Phosphate Phosphatase HAT Ac Acetylation Acetyl group HDAC OGT O-GlcNAC O-linked-Nacetylglucosamine O-GlcNAcylation OGA E1, E2, E3 Ub Ub Ubiquitination Ub Ubiquitin DUB E1, E2, E3 SUMO SUMOylation SENP, DeSI Small ubiquitinrelated modifier Phosphorylation of IRS-1 IR Y613 Y623 NH2 PH PTB PI3K binding IRS-1 has no intrinsic enzymatic activity PH – pleckstrin homology domain, binds phosphoinositides – PIPx PTB – phosphotyrosine binding domain COOH IRS-1 as a Molecular Scaffold IR Y613 Y623 NH2 PH PI3K binding PTB p85 Grb-2 COOH Insulin Signaling: Metabolism Insulin PI(3,4,5)P3 PI(4,5)P2 IRS p85 PDK1 Insulin Receptor p110 aPKC? Akt GSK3 _ GS -pS AS160 GS -pY Rab-GTP GLUT4 GLUT4 Vesicle Adapted from Fröjdö S, Vidal H, Pirola L. Biochim Biophys Acta. 2009;1792:83-92. Mitogenesis & Gene Expression Insulin PI(4,5)P2 PI(3,4,5)P3 Insulin Receptor Grb2 SOS IRS p85 p110 PDK1 Shc Akt GSK3 Erk 1/2 p70S6K mTOR Transcription Factors Nucleus Fröjdö S, Vidal H, Pirola L. Biochim Biophys Acta. 2009;1792:83-92. Insulin Action in Lipodystrophy and Type 2 Diabetes Muscle Adipose Tissue IR Binding IR Kinase IRS Phosphorylation IRS1 – PI-3K Activity Akt Phosphorylation aPKC Activity AS160 Phosphorylation GLUT4 Abundance GLUT4 Translocation GSK3 Erk 1/2 Activity Change expressed relative to healthy individuals Type 2 Diabetes Lipodystrophy PTMs of Transcription Factors: Regulation of Gene Expression Transcription Factor Modification Phosphorylation FOXO1 Effect on Activity Acetylation O-GluNAcylation Phosphorylation SREBP1c Insulin Effect Acetylation SUMOylation Phosphorylation PPARγ SUMOylation Phosphorylation Acetylation NF-кB Ubiquitination O-GluNAcylation Summary, Part 2 • Insulin is a pleotropic hormone, generating an array of responses in multiple tissues. • Insulin signaling is initiated by binding to its receptor and stimulation of IR tyrosine kinase activity. • Phosphorylation cascades regulate the assembly and subcellular localization of multi-molecular signaling complexes. Summary, Part 2, cont. • Different substrates and signaling complexes mediate insulin signaling to regulation of metabolism and gene expression/mitogenesis. • Disruption of phosphorylation cascades, either by phosphorylation at alternative sites or other PTMs, can lead to insulin resistance. Stress-Activated Kinases Kinase Substrates Activators Erk 1/2 (p44/42 Mitogen Activated Protein Kinase) C-Jun p53 IKKα Insulin Thrombin p38 Mitogen Activated Protein Kinase (p38) Caspase 3/6 SP1 CEBP FFA Glucose Inflammatory cytokines IкB Kinase (IKK) IkBα Inflammatory cytokines FFA c-Jun NH2 Terminal Kinase (JNK) IRS-1 c-Jun p53 Inflammatory cytokines FFA Phosphorylation of IRS-1 Positive Negative Mixed IR Y613 Y623 NH2 PH PI3K binding PTB S307 S312 S323 S332 S616 S636 JNK JNK GSK3 mTOR mTOR ERK S731 mTOR COOH S794 AMPK IKKß PKC PKC p70S6K p70S6K S1101 PKC Phosphorylation of IRS-1, cont. Kinase Site Effect on Activity Akt S522 , S629 JNK S307 , S312 mTOR S307 , S731 IKKß S312 PKCα S24 PKCΘ S312 , S1101 PKCζ S323 p70S6K S312 ERK S636 GSK3 S322 AMPK S794 PTMs of Transcription Factors: Regulation of Gene Expression Transcription Factor FOXO1 SREBP1c PPARγ NF-кB PTM Effect on Activity Phosphorylation Acetylation Phosphorylation Acetylation SUMOylation Phosphorylation SUMOylation Phosphorylation (IкBα) Acetylation Ubiquitination Summary, Part 3 • Kinases activated by FFAs and inflammatory cytokines can phosphorylate IRS-1 on serine residues to impair downstream insulin signaling. • Phosphorylation of transcription factors (TFs) can either stimulate or impair their activity, depending on the site modified. • Phosphorylation and other PTMs of TFs can influence subcellular localization, protein stability, and/or transcriptional activity. • Phosphorylation of IkB leads to its degradation, releasing NFkB to move into the nucleus and stimulate the transcription of proinflammatory genes, including cytokines and chemokines, maintaining a vicious circle. Hyperlipidemia and Inflammation-mediated Insulin Resistance FFA p38 DAG LCFACoA Ceramides JNK IRS IKKß Cytokines Degradation IkB NFkB NFkB Proinflammatory Genes A Downward Spiral Lipodystrophy to Insulin Resistance Ectopic Fat AT Circulating FFA Activation of Stress Kinases Phosphorylation of IRS-1, etc. Altered FA Metabolism Circulating FFA Inflammatory Cytokines Insulin Resistance Insulin Secretion
