ANAT365A Metabolism_Lecture 1 2023-1.pdf

Membranes and Cellular Trafficking: So what does this have to do with Metabolism? Dr. Jennifer Estall @dnachicken IRCM, Montréal [email protected] Outline of Lecture Lecture 1: • Why you need cellular trafficking to make and use energy properly • Hormone release from cells • Transport o...

Membranes and Cellular Trafficking: So what does this have to do with Metabolism? Dr. Jennifer Estall @dnachicken IRCM, Montréal [email protected] Outline of Lecture Lecture 1: • Why you need cellular trafficking to make and use energy properly • Hormone release from cells • Transport of energy into cells • Hormone receptor trafficking • Energy storage and transport by fat • The lipid droplet: Not just for storage! • Why lipids need to move around the cell • Viral hijacking of lipid droplets “Metabolism” Sugars § Definition: The chemical processes occurring within a living cell or organism that are necessary for the maintenance of life. § How nutrients are being used to make energy. § Many mechanisms are required to keep this complex network working efficiently – including trafficking. Fat (lipids) Protein (amino acids) Cell trafficking is an essential component of hormone processing and secretion EXAMPLE MOLECULE: INSULIN • β-cells make up 1-2% of the cells in the pancreas. • Create and secrete insulin in response to high glucose in blood. • Insulin is needed for the body to take up and store nutrients Section of the Pancreas Insulin is produced and packaged into dense core granules (DCG) in the golgi e.g. Glucose, GLP-1, KCl, sulfonylureas Kim et al. Dense Core Secretory Granule (DCG) Biogenesis, Physiology, 2009 Acidic vesicles activate proteases that cleave proinsulin to produce the active hormone PC2 (enzyme) (pH 5-6) Kim et al. Dense Core Secretory Granule Biogenesis, Physiology, 2009 Insulin is stored in vesicles distributed throughout the cell in high volumes β-cell Insulin Granule Peroxisome Golgi Mitochondria Non-β-cell (likely α-cell) nucleus Glucagon Granule Insulin Granules Mature insulin granules are only secreted in response to an external stimulus (glucose or hormone signals) Stimulus-secretion coupling pathway Ca++ channel (LTCC) Insulin release from β-cells involves highly coordinated mitochondrial and calcium signaling Mancini and Poitout, 2013 Metabolism + Trafficking are needed to control the two phases of insulin secretion • F-actin is a physical barrier. • Pre-docked vesicle release stimulated by calcium through a “stimulus-secretion” coupling pathway (1). • F-actin is reorganized by glucose entry and calcium release, allowing access of more vesicles to plasma membrane (2). • Granule recruitment to plasma membrane on microtubules supports second phase of insulin secretion (3). Caumo and Luzi, AJP – Endo & Met, 2004 Wang and Thurmond, J Cell Science, 2009 Insulin controls the entry and storage of glucose into some tissues (muscle and fat) • GLUT: glucose transporters. • Plasma membrane bound channels for glucose entry to cells. • Muscle and fat are the biggest “sink” for circulating glucose. GLUT4 GLUT4 Leto and Saltiel, Nature Reviews - MCB, 2012 Henderson and Baldwin, Nature, 2012 Glucose “sensing” and its uptake into cells is dependent on trafficking of transporters GLUT2 GLUT2 GLUT3 GLUT2 GLUT4 GLUT2 Leto and Saltiel, Nature Reviews - MCB, 2012 GLUT1 GLUT4 • Expression is tissuespecific. • Only GLUT4 is controlled by insulin – allows muscle, adipose, and heart to increase glucose entry when glucose is abundant. • Other GLUTs are not regulated in the same way, so they allow entry of glucose in other tissues at any time (important for tissues that need to “sense” [glucose]). Insulin regulates level of GLUT4 receptor at the cell surface (therefore, how much glucose can get in) Taken from lab website of Dr. David James, Garvan Institute of Medical Research, Australia • Insulin stimulates recruitment of GLUT4 to plasma membrane (10-40 fold increase) • Glucose enters through GLUT4 channel. • The level of GLUT4 on the cell membrane is directly proportional to the amount of glucose that can enter the muscle or fat cell. • Without GLUT4 translocation, you won’t get enough energy to these cells! After stimulus is gone (insulin), GLUT4 is recycled back to GSVs to wait for the next insulin burst • GLUT4 is continuously internalized by at least 2 pathways, which are tissuespecific. • GSV: GLUT4 Storage Vesicle + Insulin + Insulin • When insulin is absent, there is futile cycling of GLUT4 between endosomes and GSVs. • This prevents blood glucose from getting too low. - Insulin Leto and Saltiel, Nature Reviews - MCB, 2012 The timeline of discovery – glucose disappearance into cat muscle all the way to high resolution imaging 45 years to figure out how to measure this Another 20 to have enough data to generate a theory Identify GLUTs Technology Boom Cloning, tagged-proteins, sequencing, high res real time microscopy Stockli et al., J Cell Sci Commentary, 2011 Insulin receptor activity is tightly controlled by ligandmediated receptor internalization • Glucose uptake in muscle is very rapid and can lead to hypoglycemia (low blood sugar) if uncontrolled. Clatherin-coated pit-meditated endocytosis • Insulin action can be rapidly inactivated by internalization and degradation of the insulin receptor. • The process depends on efficient cellular trafficking machinery. Parachoniak & Park, Trend in Cell Bio., 2012 Insulin receptor internalization has purposes beyond decreasing hormone action in the cell Internalized receptor activates Ras/MAP kinase pathway to ‘mitogenic’ endpoints (cell growth/proliferation) Sustain ligand-induced Akt phosphorylation (certain targets of insulin remain “on” for a while) Andersen & Moestrup, T/BS, 2014 Ligand degradation (insulin clearance from circulation by liver – 50% of hormone) The receptor is detected in the nucleus! IR complexes may be recruited to gene loci to induce or repress transcription. First detected in 1980’s, new interest has emerged (Hancock ML Cell 2019). Levels of receptor at the cell surface control how well insulin works – implication for diabetes? High insulin pre-treatment Low insulin Pre-treatment • It is hypothesized that decreased insulin receptor levels at the cell surface contributes to insulin resistance in diabetes. • Long-term insulin exposure may lead to receptor degradation instead of recycling. • Altered recycling kinetics of the receptor seen in cells from diabetic patients and cultured cells exposed to high glucose for long periods of time. Muscle cells cultured in Low insulin (107 pM, SkMC-L) or High (1430 pM, SkMC-H) insulin concentrations. Bertacca et al. Dia Res Clin Prac, 2007 • Thought that internalization is needed to resensitize the receptor (“resetting the system”). Part 2: The Lipid Droplet – Not just for storage anymore! Human primary fat cells in culture stained for plasma membrane (red), nuclei (blue) and lipid droplets (green). Photo: Hui Gao and Niklas Mejhert. Liver tissue taken from mice fed a highfat / high fructose diet for 5 months. Photo: Estall Lab • Lipid droplets store fat in the form of cholesteryl esters or acyl-glycerols, which are fatty acids bound to a glycerol backbone (mono-, di-, and triglycerides). • Lipid synthesis and storage is stimulated by insulin • Required as a readily available source of energy during times of energy demand • All cells can store lipid, some are better than others (adipocytes, hepatocytes) Basic structure of lipid droplets and their location in the cell • • Beller et al. FEBS Letters Review, 2010 Lipid droplets have a single, phospholipid protein-containing hemimembrane (monolayer) They make physical contact with outer membranes of other organelles (shown by EM and presence of “refugee” proteins following purification). ER Mitochondria Arrese et al. Lipid Insights, 2014 Lipid droplets interact with many different organelles • Proteomic analysis of lipid droplets has identified a number of different Rab proteins that appear to regulate interactions with specific organelles. Murphy, Martin, Parton. BBA, 2008 **All lipid droplets in a cell do not all have the same associated proteins! (subsets of lipid droplets with different properties and functions) • Caveolin, a major protein component of Caveolae, may be required for lipid droplet motility. When they accumulate a dominantnegative form of the protein, they cannot move and cannot be catabolized. Formation and Movement of lipid droplets in cells • Most accepted theory is that LDs are formed following accumulation of neutral lipid between the leaflets of the phospholipid bilayers in the ER (1). ER Beller et al. FEBS Letters Review, 2010 • Free fatty acid synthesized or taken up by the cell is stored in droplet, which grows in size to accommodate. Lipases break lipid down when needed for fuel (2). Formation and Movement of lipid droplets in cells • Lipid droplets can then move around, evidenced by different locations they are found (2-9). • Intracellular trafficking machinery is tightly linked to lipid droplets (7,8,9). Their role in this is not well understood. • Form interactions with mitochondria, peroxisome and ER for exchange of proteins and lipids for fuel (4,5,6). Beller et al. FEBS Letters Review, 2010 Also consult: “Expanding Roles for Lipid Droplets” Michael A Welte, Current Biology 2017. Fat on the move: Lipid droplet movement in cells Welte, MA. Biochem. Soc. Trans, 2009 A. B. C. D. E. Mammalian cells: LDs oscillate in restricted area or travel along microtubules Chlamydia bacteria induces movement of LDs from cytoplasm into parasitophorus vacuole Hepatitis C infection causes accumulation of LDs at microtubule-organizing center LDs moving from basal to apical side of mammary-gland cells, secreted to form milk fat Dynamin-mediated LD fusion (change function?). F. Hormones (ie. adrenaline) stimulate LD fragmentation and dispersion. G. In fish eggs, LD accumulate at vegetal pole after fertilization. H. In fruit fly ovaries, LDs are produced by nurse cells and transferred to the oocyte (easy form of fuel transfer). I. As fruit fly embryo ages, predictable and coordinated LD patterns are formed. J. In fungi, LDs spread from origin near roots toward growing tip. Trafficking of lipid droplets – why is it important? 1. Storage space for newly synthesized lipids (from ER and mitochondria). 2. Recruit and carry proteins or lipid-soluble molecules to areas of cell where they are needed/used, like a cargo truck. I.e. Kinases to activate lipolysis in fat, or degradation of proteins (ERAD of ApoB), or lipophagy to eliminate misfolded proteins. ER 1. To allow fusion/fission of LDs – function of LD seems to change depending on their size and make-up. 2. Lipid droplets may serve as sites for protein translation. Ribosomes and RNA have been found on the LD surface (i.e. perilipin 1 seems to be transcribed near the LD). Viruses also use this feature to help amplify themselves (i.e. HCV)! 3. Source of lipid building blocks or fuel for organelles: allows easy transfer and use if they are in close proximity. The ER may act as a “super highway” for the lipid droplets (LDs) to move through the cell. Metabolic disease and viral infection increase the size of lipid droplets – effects on cell function? Healthy liver Hepatitis infected liver Fatty Liver The Hepatitis C virus (HCV) uses lipid machinery of liver cells to traffic within the cell and replicate • Entry: Viruses hijack cell surface receptors for lipoproteins. • Replication: Lipid droplets are scaffolds for viral assembly • Exit: Liver cells secrete lipoproteins – thus ensuring survival and spread of virus Hepatocyte (liver cell) Vieryes G et al. Viruses, 2014 Viral proteins and RNA are concentrated and transported on the surface of the lipid droplet • Viral loaded lipid droplet moves along surface of ER. • Core proteins and viral RNA are transferred from the surface of lipid droplet onto ER lumen. RNA is replicated. • Viral particle buds into ER lumen, but this is not secreted. Moriishi and Matsuura, Frontiers in Microbiology, 2012 Secreted from the cell into blood bound to VLDL particles HCV viral particles are secreted with lipoproteins, permitting infection of other cells • Viral particle is complexed with a mature lipoprotein derived from lipid droplet. • Viral infection also increases lipid production and decreases lipid droplet turnover rate – making droplets bigger. • Thus, promoting more secretion of lipoproteins (with attached virus) into circulation. VLDL: very low density lipoprotein Secreted from the cell into blood bound to VLDL particles Summary: Membrane vesicles are essential for metabolism: 1. 2. 3. 4. 5. 6. 7. Control proper packaging of hormones (dense core granules, DCG) Allow hormones to reach cell surface and be secreted (DCG) Ensure proper timing of hormone secretion (actin) Allow glucose entry in cells when needed (Gluts) Prevent glucose from entering the cell when not needed (Glut) Put the brakes on uncontrolled hormone signaling (endocytosis) Store fuel and bring these to places in the cell that they are needed (lipid droplet movement) 8. Change the activity/function of organelles (e.g. lipid droplets and mitochondrial associated membrane - MAMs) Next lecture: • Hot topics in metabolic disease • Mitochondrial associated membranes (MAMs) and nanotubules • Microvesicles and Exosomes in metabolism • Impact of cellular trafficking on the pathogenesis and treatment of metabolic disease PAPER TO READ FOR LECTURE #3: Summer Internship Program: https://www.ircm.qc.ca/en/ircm-grants-for-young-researchers