Molecular Biochemistry: Biosignaling PDF

MOLECULAR BIOCHEMISTRY BIOSIGNALING PRINCIPLES OF SIGNAL TRANSDUCTION Transduction: transmission of energy or a message between two points in a system; which implies a modification of the type of energy transmitted. Signal transduction: transmission of a signal (message) from outside the cell and...

MOLECULAR BIOCHEMISTRY BIOSIGNALING PRINCIPLES OF SIGNAL TRANSDUCTION Transduction: transmission of energy or a message between two points in a system; which implies a modification of the type of energy transmitted. Signal transduction: transmission of a signal (message) from outside the cell and its subsequent conversion into a biological response. There are two types of transductions based on the receptor:  Cell surface receptor: peptide or amine hormone binds to receptor on the outside of the cell; acts through receptor without entering the cell;  Nuclear receptor: steroid or thyroid hormone enters the cell; hormone-receptor complex acts in the nucleus; The important actors in signal transduction are receptors, they can be considered like molecular switches which can sense extracellular signals, they are responsible for initiating the response to a given signal and the response is deconvolve in to a cellular one. Receptor molecules accomplish a few important duties in the transduction process: 1. Recognize an outside signal; 2. “Transport” (it’s a virtual transport, not real) that signal across the cell's membrane; 3. Initiate the reading of the signal inside the cell and then trigger the cell's response to that signal; Some of the possible cellular responses are: secretion of molecules, changes of membrane potential (acetylcholine receptors), phosphorylation/dephosphorylation of target proteins modulating their activity (via protein kinases/phosphatase), regulation of gene function (transcription). Key features of signal transducing systems:  Specificity: signal molecule fits binding site on its complementary receptor; other signals do not fit;  Amplification: when enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade;  Modularity: proteins with multivalent affinities form diverse signaling complexes from interchangeable parts. Phosphorylation provides reversible points of interaction;  Desensitization/adaptation: receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface;  Integration: when two signal have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm, the regulatory outcome results from the integrated input from both receptors; Classification of receptors:  Gated ion channel receptors (e.g., nicotinic receptors);  Enzyme receptors (e.g., insulin receptor);  G protein coupled receptors (e.g., adrenergic receptor);  Intracellular receptor (nuclear receptors) (e.g., steroid hormone receptors);  Non enzyme receptor (e.g., erythropoietin receptor);  Adhesion receptor (e.g., integrins); 1 GATED-ION CHANNEL RECEPTORS Cellular membranes are polarized, which means that we have different charges on the two sides of the membrane: usually the positive charge is outside, while the negative one is inside. The separation of the charges is accomplished through the Na+/K+ pump, which pumps out 3 Na+ ions and pumps in 2 K+ ions  it generates differential charge across the membrane (potential). There are also other channels where other ions, like Cl- and Ca2+, enter or exit the cell generating a charge difference. Since the transport of Na+ and K+ ions is an antiport, the cell needs energy to generate the charge gradients across the membrane (ATP is needed). In the nicotinic acetylcholine receptor, there is a role of voltage-gated and ligand-gated ion channel in neural transmission. In the pre-synaptic neuron, there is an electrical signal that leads the release of acetylcholine contained in the presynaptic vesicles, once the hormone is released in the intrasynaptic space, it binds the receptor located on the membrane of the post-synaptic neuron and as result it causes the opening the channel allowing the entrance of the sodium and calcium ions that results in a subsequent cellular response of the postsynaptic neuron. The voltage gated ion channel, in this case the nicotinic acetylcholine receptor, is organized in five different subunits: two α, one β, one γ and one δ. Each subunits have a transmembrane portion that is formed by 4 alpha helixes that goes through the membrane. If we focus on the transversal section of helix M2, we can see different aminoacidic residues: bulky and non-polar residues (yellow) and small and more polar residues (light blue). The M2 helixes are facing the inside of the channel, while in the resting conditions the bulky and non-polar residues are facing the inner part of the channel blocking the entrance of the ions in the cell; 2 molecules of acetylcholine bind the two α subunits of the receptor, the binding of the molecules causes a conformational switch of the receptor: the M2 helixes of each subunits rotate and instead of exposing the bulky and non-polar aminoacid residues, they will expose the small and more polar aminoacid residues causing the opening of the channel. Also, there are aminoacid residues that can establish bonds/interactions with positive charged ions, like sodium or calcium ions. And example of a bulky amminoacid residue is leucine. The receptor doesn’t have a steady structure, but it’s a dynamic structure that can have different conformational states. The conformational switch depends on the presence or the absence of a given ligand. G PROTEIN-COUPLED RECEPTORS (GPCR) G protein-coupled receptors are also called ”7 transmembrane spanning receptors” because it has 7 alpha helixes embedded in the plasma membrane. The N-terminal portion is always extracellular, while the C-terminal portion of the protein is always intracellular. From a chemical-physical point of view the majority of aminoacids in the helixes are non-polar residues, of course there might be some polar aminoacids but they are rare. In the receptor there are many different regions that are important in each GPCR, for example some regions are important for recognizing some specific ligands so they will be different in each receptor, there’s also a region important for the interaction with the G protein (C-terminal), there’s also a region susceptible to phosphorylation that may inactivate the receptor (desensitization). 2 The G protein is formed by three subunits, it’s a heterotrimer: α, β and γ subunits. In absence of the specific ligand, the receptor is in the resting position and the α subunit of the G protein is associated with the β and γ subunits and also with GDP. In the case of the β-adrenergic receptor, when adrenaline binds the receptor, it has a conformational change that is “transmitted” to the G protein since the 2 molecules interact with each other. When the G protein changes it structure, there is a switch between GDP and GTP  it’s not a phosphorylation, is a switch: the GDP molecule is released and the GTP binds to the protein. At this point the α subunit gets activated, it disassociated from the β and γ subunits, moves across the membrane and eventually interacts with an enzyme, also localized in the plasmatic membrane, which is adenyl cyclase. This enzyme catalyzes the conversion of an ATP molecule into a cAMP molecule, cyclic AMP is defined as a second messenger which then binds and activate a kinase called PKA. PKA is a protein kinase which can phosphorylate many cellular substrates by activating or inactivating them. There is also an enzyme called cyclic nucleotide phosphodiesterase that breaks down cAMP forming AMP  the second messenger gets inactivated switching off the signal. In the case of the β-adrenergic receptor, the G protein associated is stimulatory (Gs) but there are also inhibitory kinds. Epinephrine, in general, is a hormone that prepares to action, it’s the typical hormone that is realized under conditions of stress or intense physical activity. Activation cycle of stimulatory G proteins: 3 Adenylate cyclase is a membrane protein made by several alpha helixes spanning the plasma membrane, the actual enzyme domain can be found inside the cell. Adenylate cyclase produces cyclic AMP (cAMP) from ATP: the enzyme catalyzes the released of the pyrophosphate and the formation of an ester bond between the phosphate group and the hydroxide group, localized in the 3’ position of the ribose sugar portion of the molecule, that creates a cyclic structure between C5 and C3. Then, the phosphodiesterase enzyme can hydrolyze created previously and this reaction creates AMP. AMP is an important molecule that can activate another type of protein kinase which is called AMP kinase. This enzyme is important because it’s considered a cellular sensor of the energy status of the cell: high AMP levels mean lower energy charge. In the cell there are many receptors that use cAMP as a second messenger:  Corticotropin;  Corticotropin releasing hormone;  Dopamine;  Epinephrin;  Glucagon: this hormone is released by pancreatic α-cells under low-glycemia conditions. The mistakes that most students do is that glucagon activates the β-adrenergic receptor, NO! Glucagon activates the glucagon receptor which is a different receptor that works the same way as the β-adrenergic one;  Etc.; Protein kinase A or PKA is a protein containing different subunits: A-kinase anchor protein (AKAP), 2 catalytic subunits and 2 regulatory subunits. When the PKA is not active, therefore in the absence of cAMP, the regulatory subunits are associated with the catalytic ones and because of that they are inactive. When cAMP is produced, 4 molecules bind the two regulatory subunits (2 cAMP for each unit) causing the releasing and the activation of the two catalytic subunits. cAMP activates the PKA because it causes a conformational change in the protein because it’s an allosteric modulator. 4 Lipolysis is another example of activation of β-adrenergic receptor with metabolic consequences. While doing prologue physical activity, adrenalin activates β-adrenergic receptor on the membrane of adipocytes resulting in the production of cAMP, activation of PKA that, in this kind of cell, has important effects on lipid metabolism. The PKA phosphorylates perilipin, which is a protein that surrounds lipid droplet within adipocytes, causing the detachment from CGI protein that can interact with ATGL (Adipose triglyceride lipase) which removes the first molecule of fatty acid from triacylglycerol, forming diacylglycerol. PKA also phosphorylate another lipase called HSL (hormone sensitive lipase) that removes the second molecule of fatty acid from diacylglycerol that becomes monoacylglycerol (1 molecule of fatty acid esterified to glycerol). Finally, there is a third lipase called MGL (monoacylglycerol lipase) which will hydrolyze the last molecule of fatty acid. The three fatty acids no longer esterified with glycerol are released in the bloodstream and transported to other organs (skeletal muscle, liver and cardiac muscle) where they are used as fuel to get energy. Albumin is the carrier of fatty acids in the blood stream. A typical feature of signal transduction is amplification: one signal molecule results in a more amplified final response (in the picture pay attention to the amplification factor): 5 Signal termination:  Dissociation: in this case the concentration of the ligand decreases and causes its dissociation from the receptor;  Phosphorylation: upon continues stimulation of the receptor with the ligand, the receptor kinase could be activated and phosphorylates the C-terminal portion of the receptor, this leads to a change of conformation that causes the interaction with β-arrestin leading to desensitization and inactivation of the receptor; There are several subtypes of β-adrenergic receptors: β1, β2 and β3, they are expressed on different cellular types: β2 are expressed in respiratory and cardiac system, β3 are expressed in brown adipocytes where responsible for the thermogenic response of this cell type allowing the heat production in order to control body temperature. There are others adrenergic receptor, like α2 adrenergic receptor, it transduces the signal through a G protein but the α subunit is not stimulatory, but it’s rather an inhibitory one. The mechanism is the same: upon the binding, the receptor changes the conformation, there’s the switch between of GDP and GTP, this leads to the detachment of the inhibitory α subunit that binds to adenylate cyclase inhibiting the activity. The result of α2 adrenergic receptor activation is the inhibition of the PKA-mediated cascade (opposite effect of β- adrenergic receptors). The final effect depends on the distribution of the two receptors: in tissues where the expression of α2 adrenergic receptor is higher, we will have an inhibitory effect; in other tissues where β- adrenergic receptors are more expressed there will be an activatory effect; if there’s a combination of the two receptors, the pathway depends on the concentration of the different ligand. α2 adrenergic receptor causes:  Suppression of release of norepinephrine by negative feedback;  Transient hypertension, followed by a sustained hypotension;  Vasoconstriction of coronary artery; however, the extent of this effect may be limited by the vasodilatory effect from β2 receptors;  Constriction of some vascular smooth muscle;  Decrease motility of smooth muscle in gastrointestinal tract;  Inhibition of lipolysis;  Sedation and analgesia;  Inhibition of insulin release in pancreas;  Induction of glucagon release from pancreas; There are some bacterial toxins that may affect the G protein’s activity:  Cholera toxin: introduces ADP-reboxy on arginine on the G2 domain of the alpha subunit of the Gs protein. The toxin blocks the intrinsic GTPase activity and α remains in a state of constant activation;  Pertussis toxin: toxin introduces an ADP-reborose on a cysteine in the C-terminal region of Gi/G0 proteins. Interaction with the receptor is prevented and there is no activation of these systems; β and γ subunits have also an important role, because the complex prevents the activation of the α-GDP subunit when the receptor is not activated by the agonist (regulator of α subunit activity), when the three subunits are together, the α subunit cannot be activated, it prevents the constituent activity; when the receptor is activated by the agonist, it binds the αβγ heterotrimer with high affinity; β and γ contribute to receptor desensitization: they stimulate β-adrenergic kinase (βARK) which phosphorylate the C-terminal portion of the receptor leading to its inactivation; in some cases, it has been showed that β and γ subunits may contribute signal transduction by interacting with effectors (e.g., phospholipase C, Src-like protein, PI3-K, Ras). Desensitization of β-adrenergic receptor upon prolonged exposure to epinephrin: 1. Upon a continues stimulation by the ligand, the C-terminal portion gets phosphorylated by βARK; 6  2. As a consequence, the receptor gets associated with β-arrrestin proteins; 3. β-arrrestin proteins lead to the formation of a structure that causes the endocytosis in vesicles of receptor molecules which are no longer exposed on the cell surface; 4. Since there is no receptor on the surfaces, it cannot be activated by the ligand; 5. Only when the receptor gets dephosphorylated in the vesicle, this can migrate back to the cellular membrane allowing the activation by the ligand; NB: PKA can phosphorylate and activate βARK  this is an autoregulatory loop (negative feedback), the receptor can control its own activity. Recently it has been also described another effect of β-arrrestin which is mediated by an endosome containing Rab5 protein: β-arrrestin lead to the receptor internalization, once the complex is in the endosome vesicles, this may lead to the activation and production of cAMP in the endosome. For example, the α stimulatory subunit can regulate glucagon-like peptide receptor that is activated by the glucagon-like peptide which is an important peptide that regulates glucagon homeostasis. The receptor may mediate the generation of Rab5 endosomes, which lead to the activation in the endosomes of adenylyl cyclase. Let’s consider another signaling cascade that uses GPCR. This signaling pathway is mediated by a different second messenger: the downstream effector, in this case, is phospholipase C. Phospholipase C is a phospholipase located in the plasma membrane which can breakdown PIP2 in two products: diacylglycerol (DAG) and inositol 1,4,5-triphopshate (IP3), in particular it catalyzes the hydrolysis of the ester bond. The G protein used in this pathway contains the Gqα subunit, that interacts with phospholipase C which becomes activated and hydrolyzes PIP2. The downstream effect is that diacylglycerol activates the protein kinase C (PKC) and then can phosphorylate other substrates within the cell regulating their activity; IP3, will bind calcium channels located in intracellular membranes (endoplasmic reticulum) and will open them causing the releasing the stored calcium in the cytosol, this increase contributes to activate some members of the PKC family. Another protein that can be activated by calcium is calcium-calmodulin, which then activates calcium-calmodulin kinase that phosphorylate other substrates within the cell. The receptors the use this pathway are α1-adrenergic receptors: they primarily mediate smooth muscle contraction, but have important functions elsewhere as well. The neurotransmitter norepinephrine has higher affinity for the α1 receptor than does the hormone epinephrine/adrenaline. TYROSINE KINASE RECEPTORS (INSULINE SIGNALING) Insulin receptors belong to the family of tyrosine kinase receptors, they are receptors which have intrinsic tyrosine kinase activity: there’s a domain that catalyzes the phosphorylation of tyrosine residues on target proteins. Insulin timeline:  1550BC: first report of diabetes cases. An Egyptian papyrus describes symptoms compatible with diabetes;  1910: insulin deficiency is identified as the cause of diabetes;  1921: Banting and Best extract insulin. Insulin extract from a dog’s pancreas is injected to treat another dog with diabetes ad Macleod’s laboratory at the University of Toronto;  1923: Banting and Macleod are awarded the Nobel Prize for the discovery of insulin;  1958: Frederick Sanger is awarded the Noble Prize for the structure of insulin. Insulin becomes the first protein to be sequenced;  1982: the first synthetic human insulin is commercialized as Humulin; 7 Insulin is produced by β-cells in the endocrine pancreas, where there are the so-called Langerhans Islet where we can distinguish different cell types:  α-cells important for glucagon production;  β-cells important for insulin production;  δ-cells important for somatostatin production;  Exocrine cells important for hydrolytic enzyme production, enzymes important for protein digestion; Metabolic effects of insulin: Insulin is synthesized and pre-pro-insulin that contain the N-terminal signal sequences, which is then removed creating the pro-insulin that contains the S bonds between cysteine residues, another step is the removal of the so-called C peptide, eventually we end up with the mature insulin that contains two peptides: α- and β-chain kept together by the cystine bonds (disulphide bridges), the final molecular weight is 58’000Da. Peptide C of insulin in considered an important marker of insulin secretion and β-activity, it’s a diagnostic tool that allows to discriminate between type 1 and type 2 diabetes (indicator of pancreatic activity). Type 1 diabetes is due to failure of β-cells which are no longer capable of producing insulin, it’s an early on-set disease; type 2 diabetes is due to the fact that pancreas is still capable of producing and secreting insulin, but target tissues are no longer capable to respond properly, these tissues become insulin resistant. It seems, from some reports, that peptide C can bind to receptors (GPCR?) of endothelial cells, neurons, fibroblast and renal tubular cells, this could lead to the activation of calcium-dependent signaling cascade (PLC, KC, MAPK); there are some reports that show that administration of peptide C in animal models affected by type 1 diabetes can improve diabetic neuropathy and renal function. Why can the C-peptide assay be useful? The dosage of C-peptide levels in the blood is useful to estimate the production of endogenous insulin by the beta cells of the pancreas. If blood levels of C-peptide are low, presumably insulin synthesis is also poor. From a clinical point of view, this parameter is very useful both to 8 better define the characteristics of newly diagnosed diabetes and to verify over time the residual capacity of insulin production in long-standing diabetes. How can it vary in subjects with type 1 and type 2 diabetes? For what has been said so far, the dosage of C- peptide will naturally be more or less reduced in subjects with type 1 diabetes, in which insulin secretion is partially or totally impaired. According to the Italian Society of Diabetology (SID), based on the scientific literature, reduced C-peptide levels have clinical significance and appear useful to characterize subjects at higher risk of rapid disease progression, chronic complications, worse metabolic control and severe hypoglycemia. The most recent studies have documented that in some subjects, C-peptide production persists, particularly in subjects with onset of diabetes in adulthood. The presence of stable levels of C-peptide even after decades of diabetes is associated with a more favorable clinical profile in terms of disease control and prevalence of complications. The dosage of C-peptide is also useful in subjects with type 2 diabetes in which the value can be extremely variable on the basis of the natural history (= evolution) of the disease, which varies from subject to subject, and is able to guide the therapeutic choices, especially with regard to the need to use exogenous insulin in cases of type 2 diabetes in advanced stages, against values of C-peptide particularly low and therefore indicative of pancreatic insulin stores (endogenous insulin) in exhaustion. Why is the blood dosage of C-peptide preferred and not of circulating insulin (insulinemia) to assess pancreatic beta cell function? There are several reasons. First, the rate of degradation of C-peptide is slower than that of insulin (half-life of 20-30 minutes versus 3-5 minutes), which allows for more stable values. In healthy subjects, the fasting plasma concentration of C-peptide is around 0.3-0.6 nmol/l, with a postprandial increase of 1-3 nmol/l. Second aspect: insulin secreted by the pancreas is predominantly metabolized in the liver while C- peptide is catabolized renally. The clearance rate is constant for C-peptide but not for the hormone. Last but not least, C-peptide does not react (unlike the hormone) with anti-insulin antibodies that may form in people with diabetes under insulin therapy. The determination of C-peptide allows therefore to measure the rate of endogenous insulin, that is produced by the body, even in case of exogenous insulin administration (injections in patients with diabetes) or in the presence of anti-insulin antibodies that interfere with the dosage of the hormone. The C peptide assay is also useful as an aid in the diagnosis of insulinoma, a tumor of the insulin- secreting beta cells of the pancreas. Insulin secretion: 1. Upon ingestion of a meal containing carbohydrates (glucose), the high concentration of glucose in the blood leads to the internalization in β-cells through GLUT2 transporter. GLUT2 is a transporter with low glucose-affinity, meaning that it will can be capable of its internalization under postprandial condition (after a meal)  high glycemia; 2. GLUT2 internalizes glucose which undergoes glycolysis (pancreatic glucokinase, also has low glucose- affinity); 3. Because of glycolysis and Kreb’s cycles the production of ATP increases; 4. High concentration of ATP in β-cells blocks the potassium channel preventing the exit of potassium ions outside the cell. This leads to the depolarization of the β-cell’s plasma membrane; 5. The depolarization is responsible for the activation of the voltage-gated calcium channel which opens, calcium enters and causes the releasing of insulin from insulin-containing granules; In the potassium channel there are several subunits, in particular the activity of SUR1 subunit can be modulated by different molecules some of them are physiological others are pharmacological agents. ATP is an inhibitor; therefore, it determines the closure of the channel followed by the depolarization of the membrane, stimulating insulin release; vice versa, ADP and PIP2 stimulates the opening of the channel inhibiting insulin release. Tolbutamide belongs to a family of drugs (sulphonylureas) used as anti-diabetic agents for type 2 diabetes, this molecule is able to increase the production of insulin by pancreatic β-cells. In type 2 diabetes, a way to compensate the insulin-resisting phenotype is to produce more insulin: in the early stages we cannot observe 9 macroscopic disturbances because the glucose level may be normal and our body is responding to a low sensibility by producing more insulin. Insulin receptor is made by 2 α subunits, exposed to the outside, and 2 β subunits, embedded in the plasma membrane and have an intracellular portion that contains the tyrosine kinase activity, these subunits are kept together by cystine bonds. Insulin receptor contain intrinsic tyrosine catalytic activity, it means that it can phosphorylate tyrosine residues. 1. In the inactive form, the receptors are in monomeric form and the tyrosine kinase domain is inactive; 2. When the ligand binds the receptor, the binding induces a conformational rearrangement and the receptors dimerize; 3. Dimerization allows the transphosphorylation of catalytic domains, called activation lip, so each catalytic domain phosphorylates the receptor domain next to it to make it strongly active; 4. Phosphorylation generates a conformational change by making the active site accessible thanks to an insertion of negative charges that make lip move away from the active site; 5. Receptors phosphorylate tyrosine residues of downstream targets like Inulin Receptor Substrate (IRS); 6. The activation of IRS leads to the recruitment of phosphoinositide 3-kinase (PI3K); 7. PI3K will phosphorylate PIP2 leading to the formation of PIP3; 8. PIP3 can recruit another protein kinase called PDK1; 9. One of the targets of PDK1 (PIP3-depentent protein kinase) is another protein kinase called Akt; 10. Akt, upon phosphorylation by PDK1, becomes active and therefore can phosphorylate a number of downstream targets in the cell; Insulin receptor is not the only member of the tyrosine-kinase receptor family, there are other members like: vascular endothelial grow factor receptor, platelet derived grow factor receptor, epidermal grow factor receptor, nerve grow factor receptor, fibroblast grow factor receptor, etc. Insulin can also signal through a different pathway: MAP kinase pathway. 1. Insulin receptor binds insulin and undergoes autophosphorylation on its C-terminal tyrosine residues; 2. Insulin receptor phosphorylates IRS-1 on its tyrosine residues; 3. IRS-1, when phosphorylated, can recruit Grb2 and Sos proteins which are modular proteins that can recruit Ras protein (a small G protein); 4. Ras, when it’s not activated is associated to GDP, upon recruitment by Grb2 and Sos proteins will release GDP and bind GTP; 5. Ras becomes active and will recruit Raf1, which is a protein kinase that phosphorylates MEK; 6. MEK, upon phosphorylation, phosphorylation another protein called ERK; 7. ERK enters the nucleus and phosphorylates Elk1 (transcription factor); 8. Phosphorylated Elk1 joins SRP to stimulate the transcription and translation of genes needed for cell division; One important function of insulin is regulating glycogen synthesis by stimulating the activity of glycogen synthase: 1. Insulin activates the receptor; 2. The tyrosine kinase activity phosphorylates IRS-1; 3. IRS-1 activates PI3K; 4. PI3K phosphorylates PIP2 in PIP3; 5. PIP3 recruits PDK1; 6. PDK1 activates Akt; 10  7.Akt phosphorylates GSK3, which is glycogen synthase kinase 3, and when phosphorylated becomes inactive. When not phosphorylated GSK3, it phosphorylates glycogen synthase causing an activity decrease;  This means that when there is insulin, Akt phosphorylates GSK3 causing its inactivation which means that the activity of glycogen synthase increase causing the glycogen formation. Another important effect of insulin is the stimulation of GLUT4: insulin activates the receptor which lead to Akt activation; Akt activation leads to the translocation of intracellular vesicles containing GLUT4, which will be exposed to the plasma membrane and will be available for internalize glucose from the blood. There is crosstalk between insulin receptor cascade and β-adrenergic receptor cascade. In particular, insulin activates the receptor which lead to Akt activation; Akt and the tyrosine kinase portion of the receptor can phosphorylate some residues in the C-terminal portion of the β-adrenergic receptor leading to its inactivation through the β-arrestin system. This a way to modulate other intracellular cascade by different receptor signaling mechanism. Another possibility is that the tyrosine kinase activity of the receptor can phosphorylate other residues of other different GPCR (no β-adrenergic receptor); this may lead to recruitment of the MAPK cascade that has effects on gene expression. Insulin signaling can be modulated in physiological and pathological conditions. In case of physiological conditions, we may have other types of phosphorylation on IRS-1, under these conditions the molecule will be much less active; therefore, this is a way to downregulate the activity of the insulin receptor cascade (this is a negative feedback loop, a way to turn off insulin signaling). However, there are conditions under which the kinase that phosphorylate IRS-1 are hyper stimulated and therefore they cause a pathological condition induced by proinflammatory cytokines (TNFα, IL-6, IL-1 β) or by excessive amount of free fatty acid accumulating in skeletal muscle or in hepatocytes  leads to insulin resistance. How we become insulin resistant? Under overweight or obesity conditions, adipocytes become bigger and they tend to release proinflammatory cytokines and non-esterified fatty acid, these factors may hit skeleton muscle fibers or hepatocyte and cause a number of negative consequences, for example proinflammatory cytokines will activate protein kinases leading to reduced insulin sensitivity; free non-esterified fatty acid can accumulate and they activate the kinases causing the inactivation of insulin signaling. Inflammasome and insulin resistance in aging cell: it is well-documented that the rate of aging can be slowed, but it remains unclear to which extent aging-associated conditions can be reversed. Understanding the reversibility of aging-associated conditions has important implications in treating aging-related diseases. Here, researchers at the University of California, Berkeley discovered that the SIRT2 enzyme regulates the NLRP3 inflammasome, a cellular machinery that produces inflammatory cytokines and prevents inflammation and insulin resistance in aged but not young mice. The researchers demonstrated that SIRT2 and NLRP3 modulation can be targeted to reverse inflammation and insulin resistance in aged mice. These findings demonstrate an underlying cause of aging associated inflammation and highlight the reversibility of aging-associated conditions. Insulin is produced in and secreted from pancreatic β-cells, but it has been unclear how insulin signaling is regulated in these cells. Ansarullah et al. have discovered a previously unknown regulator of insulin signaling in β-cells, the protein inceptor. The group finds that inceptor binds to pAP2M1, a subunit of the AP2 protein complex. This triggers a process called clathrin-mediated endocytosis, in which inceptor and insulin receptors (along with the related insulin-like growth factor 1 receptors, not shown) are engulfed by the cell membrane and enter the cell. Insulin therefore cannot bind to its receptor. This insulin desensitization restrains insulin signaling to fine-tune insulin secretion from, and proliferation of, β-cells, maintaining normal responses to glucose. b, Deletion of inceptor prevents internalization of insulin receptors through this pathway, thereby allowing unrestrained insulin action and leading to enhanced insulin secretion and an increase in β-cell proliferation. These findings identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy. 11 Insulin regulates glycolysis which is mediated through an important reaction mediated by phosphofructokinase-1 (PFK1) that catalyzes the formation of fructose 1,6-biphosphate starting from fructose 6-phosphate. PFK1 is an allosteric enzyme which is positively regulated by fructose 2,6-biphosfate that, at the same time, negatively regulates the enzyme called fructose bisphosphatese-1 (catalyzes the conversion of fructose 1,6-biphosphate in fructose 6-phosphate, gluconeogenesis). The key component is fructose 2,6- biphosfate, that is produced by PFK2: a bifunctional enzyme. PFK2 contains 2 catalytic activities: kinase and phosphatase activity, the first one leads to the formation of fructose 2,6-biphosfate, whereas the second one leads to the disruption of fructose 2,6-biphosfate forming fructose 6-phosphate. Insulin is a positive regulator of the PFK2 kinase activity, in this case the phosphate group is removed; on the other end, glucagon, through the GPCR receptor, phosphorylates PFK2 causing the increase of the phosphatase activity. β-adrenergic receptors are also present on a particular type of adipocytes  brown adipocyte, which are capable of producing heat that is important for maintaining body temperature in mammals. Norepinephrine is released from hypothalamic regions when we are exposed to cold, the hormone binds β3-adrenergic receptors on brown or beige adipocyte (which are white adipocyte that, under some condition, can be similar to brown adipocyte). The receptor transduces the signal thorough a G stimulatory protein causing the activation of PKA, this leads to the activation of lipolytic pathway which causes the activation of Ucp1 (Uncoupling Protein-1 or thermogenin). Ucp1 is located in the inner mitochondrial membrane and it’s basically a proton channel. Upon stimulation of Ucp1 by free fatty acids, the channel opens up causing the entering of protons in mitochondria matrix, the final consequence is the dissipation of the proton gradient, which builds up as a result of the electron transport chain that is used to synthesize ATP. The energy of the gradient is dissipated as heat. Another signaling molecule is leptin, which is produced in white adipose tissue after ingesting a meal. This molecule has an important role controlling food consumption because when it’s released in the blood, it binds to special hypothalamic neuros that release signal which control food consumption (stop eating). This is what happen in normal people, in overweight and obese subjects the signaling pathway is dysfunctional, these subjects are leptin-resistant. Therefore, this is why these individuals do not have a proper food consumption. When we eat too much food containing fat, fatty acids and fat derivatives can accumulate in different organs and tissues and also in the neurons caring the receptor for leptin and therefore the receptor doesn’t respond well to the molecule and consequently the subjects become leptin-resistant. 12 NON-ENZYME RECEPTORS Leptin works using another type of receptor: non-enzyme receptors, they don’t have catalytic activity but when they are activated by the ligand, in this case leptin, the associates to the Janus Kinase (JAK), which phosphorylates the leptin receptor that recruits STAT (transcription factor, Signal Transducers and Activator of Transcription). STAT, as a consequence, becomes phosphorylated itself and migrate into the nucleus where stimulates the transcription of specific set of genes, among which there are peptides involved in the control of food consumption (e.g., POMPC which is an anorexigenic peptide, it decreases hunger). GENE TRANSCRIPTION Through transcription, information in the DNA is converted in RNA. Since gene transcription is a process finely regulated, it is linked to signaling pathways. For example, some hormones can activate a special transcription factor called CREB (cAMP Response Element Binding protein): this factor is activated upon phosphorylation mediated by PKA and one of its targets is the gene encoding for Ucp1. There are different levels where regulation of a specific function can occur:  Transcription initiation;  Elongation of nascent mRNA;  RNA splicing;  Export of RNA in the cytosol;  RNA turnover (half-life);  Translation of RNA in a protein;  Protein turnover (half-life);  Posttranscriptional modifications;  Allosteric regulation; The regulation of gene expression has fundamental implications in biology:  Differentiation: there are genes important in regulating the differentiation of, for example, stem cells to a specialize cell type;  Development: individuals, in their life, go through different developmental processes which are regulated at the level of gene transcription;  Regulation of metabolism: many metabolic pathways are regulated at the level of gene transcription. Example: gluconeogenesis, one of the main enzymes of this pathway is phosphoenolpyruvate carboxy- kinase (PEPCK) which is regulated at transcriptional level  by regulating the gene encoding the enzyme, extracellular signals such as insulin and glucagon can regulate gluconeogenesis;  Pathology: if any of these processes is not properly regulated, a state of disease can occur. Example: PEPCK; in diabetes, the insulin signal doesn’t properly, PEPCK is not shut down and so, after ingesting a meal, the level of glucose increases in the bloodstream but in the same time the production of glucose in the liver doesn’t stop and this contribute to build up hyperglycemia that become pathological; Transcription means reading of generic information int the DNA and conversion into RNA, the enzymes involved are RNA polymerases (there are three different types, in case of mRNA type 2 is involved). Not all genes are expressed at the same time, the transcription profile changes at different stages of development and differentiation, each cell type/tissue transcribes and expresses a precise set of genes (this is way there are differences). PROMOTERS In transcription regulation there are different elements involved in genetic information, the most important is called promoter. A promoter consists in sets of DNA sequences (so-called responsive elements) and their 13 associated factors that control gene expression. All DNA are oriented relative to the 5’ transcriptional side, defining 5’ end of the mRNA is a key factor in transcriptional regulation. A generic promoter is formed by an upstream activating sequence (UAS) and the TATA box, which is the site where the RNA polymerase and all the associated protein bind. This sequence is called like that because it is enriched of thymine and adenosine residues which are characterized by only two hydrogen bonds, meaning that the DNA in this region can open more easily and this is important in the formation of the so-called “transcription bubble” where the RNA polymerase2 + the many other protein components of the transcriptional machinery can bind and move along the DNA and transcribe  the duple helix opens in order to facilitate the transcription. The UAS sequence is also called enhancer which usually located far away from the transcription start site (TSS, it corresponds to the first nucleotide that will be transcribe into mRNA) of a given gene, it can be upstream or downstream the coding region. RECRUITMENT The key for transcription is the word “recruitment”: the capacity to aggregate many different proteins, forming a complex, which is activated and can initiate the transcription process. In the picture, different transcription factors are represented: TFIID first binds the TATA box and this is the platform that recruits other general transcription factor which then bind to the promoter and then the RNA polymerase 2, but before starting the process it needs something that helps the activation  TFIIH, a general transcription factor that is involve in the activation of the enzyme. For example, TFIID is not a single polypeptide, but it is a complex that contains many different polypeptides like TATA binding protein and TATA associating factor. The large complex that forms by the binding of the different transcription factors is called pre- initiation complex (PIC). After PIC is formed, RNA polymerase 2 can start the transcription process: the duple helix opens, the transcription bubble opens and the transcription can start. The C-terminal portion of the largest subunit of RNA polymerase 2 contains several many repeats (max 50 times) of an heptapeptide: tyr-ser-pro-thr-ser-pro-ser. Serine 2 and 5 residues are a phosphorylation target that is important for the activation of the polymerase. CDK-7 and CDK-9 are protein kinases that phosphorylate the serine residues; in particular, CDK-7 phosphorylations on ser-5 are important for promotor clearance (it helps the release of the complex and the beginning of the transcription), CDK-9 phosphorylations on ser-2 are important for the elongation of the nascent mRNA, otherwise we would have a spurious transcription with short RNA which will not be the right length. In order to have an active transcription machinery we also need other molecules such as transcription activators, for example CREB, which somehow “communicate” with PIC leading to the activation of the transcription of target genes. The preinitiation complex alone is not enough. Transcription activators are important because they can bind specific sequences of the DNA, which can be close or far from the core promoter. These factors communicate with PIC by interacting with the so-called mediator complex which then recruits the general transcription factors machinery + RNA polymerase 2. In complex organisms, like mammals, we can have different transcriptional activators each of which can sense different extracellular signals  the final effect on the transcription of a specific gene is the result of the combinatorial action of different transcription factors. 14 In general, promoter sequences are located between nucleotide -40 and +50 relative to TSS, it binds and assembles the preinitiation complex, determines transcription initiation and it’s regulated by activators and repressors. PIC contains RNA polymerase 2, general transcription factors (TFIIs) and coactivators and corepressors (coregulators). Coregulators are the proteins that help the communication between the transcriptional activator and the general transcription machinery (PIC). Transcription activators (or factors) are proteins containing different functional modules:  DBD: DNA Binding Domain, portion that represents the interface with the DNA. It’s called also response element;  SSD: Signal Sensing Domain, this region can sense extracellular signals, there are different ways to do so: direct bond (e.g., estrogens bind to estrogen receptors) or post translation modification of SSD leading to activation or repression of the transcription activator;  TAD: Transcription Activation Domain, region responsible of activation of the transcription activator; More transcription factors bind consensus sequences regulating a given gene: combinatorial control of transcription. Combinatorial control: Different activators interact with different portion of the machinery and therefore will lead to a combine action in to the final transcription rate, which can be higher or lower depending on which transcription factors are recruited and which of them is more or less active. 15 CO-ACTIVATOR CBP CBP stands for CREB Binding Protein, the name comes from the initial identification of this transcriptional activator which was originally identified as a co-activator of the transcription factor CREB. Co-activators and co- repressors do not bind the DNA directly; in particular CBP can interact with nuclear receptors, transcription factors such as Fos, Jun, CREB, p91 etc. Different transcription factors sense different extracellular signal like growth factors, peptide hormones, ligand, etc. We may have synergies between these transcription factors which may bind in a combinatorial manner. The transcription of a given gene is the result of the integration of different signals, for example the transcription of PEPCK gene is positively regulated by glucagon and glucocorticoid, while is negatively regulated by insulin. GENE TRANSCRIPTION IN EUKARIOTES In higher eukaryotes, the DNA is not naked but it’s associated to nucleosome forming a super-compacted structure called chromatin. Chromatin structure is fundamental in regulating the transcriptional activity of different genomic regions. We may have genomic regions with a structure allowing the transcription but we also have regions not favoring the transcription. The different position of nucleosome may result in different capacity in recruiting or not transcription activators: There are two possible situations:  Chromatin is more compact (heterochromatin): the DNA is tightly bound to nucleosome, under this condition we find transcription factors associated to proteins called histone deacetylases (HDAC), which are epigenome modifiers. HDACs remove acetyl group from histone tale and therefore they favor the compaction of chromatin  acetyl group are associated to lysine residues, therefore if we remove 16  those groups the positive charge of the residue is free and so the chromatin structure gets more compact;  Chromatin is loose (euchromatin): the lysine residues get acetylated and therefore there is no more positive charge attraction and therefore we open chromatin which is more accessible to transcription factors; The balance of acetylation or deacetylation is the result of the balance of the activity of histone acetylates, activated by co-activators and histone deacetylases, activated by co-repressors. Among HATs we have CBP and p300 (300 is the molecular weight), which form complexes with P/CAF. These proteins do not only acetylate histones but can also acetylate other general transcription factors. HDACs associate with corepressors like SMRT/N-CoR, they can also acetylate other proteins as well like p53, HNF-4, etc. RNA editing is a simple process whereby a gene can code for two different proteins for example apolipoprotein B100 (found in LDLs, 100 stands for 100% of the protein) and B48 (found in chylomicrons, 48 stands for 48% of B100). These two proteins come from the same gene, but while B100 is a long protein, B48 is much shorter: in the liver there is the sequence of mRNA that contains the sequence CAA which codifies for glutamine causing the synthesis of apolipoprotein B100; in the intestine there is an enzyme called cytidine deaminase that converts the cytosine in uracil which causes the formation of a stop codon that results in a shorter protein: apolipoprotein B48. TRANSCRIPTION FACTORS As we already anticipated, transcriptional activators are nuclear proteins, also called transcription factors, that bind sequences located nearby, or far from (in this case they bind to enhancer elements), the core promoter and contain at least three functional domains: DNA binding domain (DBD), signal sensing domain (SSD) and transcription activation domain (TAD). Role of liver-enriched transcription factors in hepatic gene expression At the beginning these factors were found in the liver, so the researcher thought they were only expressed there but they are not since they are expressed in adipocytes, other organs as well.  HNF-1: is a transcriptional regulator forming both homo- and heterodimers of the two isoforms HNF- 1α and HNF-1β, two distinct gene products with different properties of transcription activation. Only HNF-1α is expressed in the liver, whereas 1β is expressed in other tissues and cell types;  HNF-3: this subfamily belongs to FOX (forkead box) family of transcription factors and include at least 30 members and is express in a wide variety of cell types. HNF-3α, -β and -γ bind as monomers to the same DNA consensus sequence;  HNF-4: belongs to the nuclear receptor superfamily. Subtype α is expressed in the liver, kidney and gastrointestinal tract, subtype β and γ in the kidney, pancreas, duodenum, colon, testes;  HNF-6;  C/EBP; CREB CREB stands for cAMP-responsive element binding protein, it is activated by cascades elicited by a given agonist acting on G protein-coupled receptors associated with G stimulatory proteins which activate the PKA module. In this case, one of the many target proteins of PKA is CREB; in particular, PKA phosphorylates and activates CREB by phosphorylation a serin residue in position 133. Upon phosphorylation, CREB can recruit co-activators, like CBP which has intrinsic histone acetyl-transferase active and contributes to the activation of a specific gene transcription and also to chromatin opening. 17 CREB contains different functional domains:  KID domain (Kinase Inducible Domain): it is called this way because contains some amino acid residues which can be phosphorylated and their phosphorylation can modulate the transcriptional activity of CREB. Ser133 is phosphorylated by a number of basic directed kinases including protein kinase A (PKA) and PKC. Two clusters of phosphorylation sites flanking Ser133 inhibit CBP/p300 binding. Ser residues at these sites are phosphorylated by various kinases, including ataxia-telangiectasia mutated (ATM) at Ser111 and Ser121 and calcium- and calmodulin-dependent kinase II (CaMKII) at Ser142;  Q1 and Q2: in particular Q2 is important for the interaction with the component of the general transcription factor TFIID which is responsible of interacting with the TATA box, it contributes to the formation of PIC. TFIID is a large protein containing different protein subunits, such as TIB and TAF; in particular Q2 is the portion of CREB that interacts with TAF4;  bZIP (basic leucine zipper): is a typical motive in some transcription factor which is found in the DNA binding domain, in particular this structure is important for recognize some specific DNA sequences (consensus sequence). This portion is also important for the nuclear localization of CREB; Gluconeogenesis is a metabolic pathway activated during fasting condition and physical activity that generates glucose in order to raise glycemia in the bloodstream. The most important reaction in the pathway is the one catalyzed by the PEPCK enzyme: The cytosolic form of PEPCK (there is also a mitochondrial form) is regulated through a very complicated series of mechanisms, the key to understand the regulation is the transcription factor FOXO1 the key to understand the regulations is FOXO1 and the co-activators interacting with it and also the nuclear receptor HNF-4, at least in liver. PEPCK is also expressed in adipocytes but plays a role in a different metabolic pathway: glicerolneogenesis, instead of producing glucose this pathway generates glycerol 3-phosphate, which is required for esterification of fatty acid into triglycerides. The region of the DNA that contains binding sites for different transcription factors and nuclear receptors is called hormone responsive unit (HRU) because it contains the information to regulate transcription in response to different hormonal signals. How is PEPCK regulated in hepatocytes? One of the key transcription factors that bind to the HRU is FOXO1, that, like all transcription factors, needs co-activators in order to be active and promote transcription of target genes. The co-activator, in this case, is PGC-1. The transcription of PEPCK, and therefore gluconeogenesis, is positively regulated by hormonal signals that are typically produced during the fastest state or while physical activity: epinephrine and glucagon  these hormones activate the PKA leading to the activation of CREB which binds to CRE (DNA consensus sequence for CREB) and activates the transcription of co-activators like PGC-1. Vice-versa, after ingesting a meal, insulin is secreted and one of its many effects, in the liver, is the repress of FOX01, therefore the transcription of PEPCK is reduced. 18 PGC-1 stands for PPAR gamma coactivator 1α and it’s a transcriptional activator which was discovered at the end of the 90s by some researcher that gave the name simply because it was the co-activator of the nuclear receptor PPAR gamma. But, like it always happens, PCG-1 is not only the specific activator for PPAR gamma but it can bind other transcription factors. The activation of insulin receptor leads to the activation Akt (or PKB) phosphorylates FOX01 causing its degradation by the proteasome. In addition to glucagon and epinephrine, glucocorticoid hormones are released during fasting condition and physical activity and causes the transcription of the PEPCK gene. SREBP-1C SREBP-1c, SREBP stands for sterol regulatory element binding protein 1 (there is also type 2), is a transcription factor that regulates the transcription of lipogenic genes (genes encoding enzymes involved in the novel fatty acid synthesis, for example acetil-CoA carboxylase and fatty acid synthase). SREBP-2 is mainly involved in the regulation of genes related to cholesterol synthesis and transport (for example, LDL receptor). It has been showed that insulin can activate the transcription of the gene encoding SREBP-1c, so if we increase its production, we will expect an increase of fatty acid synthesis  insulin is lipogenic; this is why in diabetes we don’t have to focus only on glucose metabolism, but we also have to pay attention to metabolic pathways. Insulin activates SREBP-1c in liver at two levels:  It increases SREBP-1c transcription in the final protein;  it increases transcription of the SREBP-1c gene, leading to increased SREBP- 1c mRNA and precursor protein; This transcription factor has been discovered in the early 90s by two Nobel prizes and was named after a stereo regulatory element binding protein. SREBPs are a very strange and peculiar type of transcription factor: when they are synthesized and not properly activated, they are not in the nucleus but they are placed in the endoplasmic reticulum membrane where they are associated to other proteins called SCAP (SREBP cleavage activating protein) which are associated to another protein called Insig (insulin induced gene) and its function is to prevent the migration of SREBP in the nucleus thanks to some cholesterol derivates such as oxysterol and sterol. When the intracellular level of cholesterol and its derivates decreases, Insig is degraded and consequently SCAP will allow the movement, along with another protein called Sec, of SREBP toward the Golgi apparatus where there are two proteases, site 1 and site 2, that cleave SREBP and the N-terminal portion will be released and moves to the nucleus where activates the transcription of lipid-synthetizing enzymes. Insulin, in this process, has the role to facilitate the cleavage and the translocation of the N-terminal portion in the nucleus. 19 CHREBP ChREBP stands for Carbohydrate response element binding protein, which is a lipogen transcription factor which is able to activate transcription of genes in lipogenesis directly sensing glucose level within cell (thank to xylulose-5P, which is a molecule that comes from the pentose phosphate pathway, active after ingesting carbohydrate containing meal), without the direct role of insulin. Under fasten condition, we have low level of glucose in the blood, low level of insulin and therefore we have higher level of glucagon that causes the activation of cAMP/PKA cascade. As a consequence, the activation of PKA leads to the phosphorylation of ChCREBP and so it cannot migrate to the nucleus where can’t activate the transcription of lipogenic and glycolytic genes in the liver. After a meal, we have low glucagon level and high insulin level but most importantly we have high glucose level in both bloodstream and hepatocytes which results in the production of xylulose-5P activates some protein phosphatase, called PP2A, which dephosphorylate ChREBP. Dephosphorylated ChREBP is more active and is able to move to the nucleus where it can bind the consensus sequence and promotes the transcription of target genes. There is a double dephosphorylation: one in the cytosol and one in the nucleus. In particular, in the nucleus, ChREBP heterodimerizes with Mlx1 and the heterodimer will be the active transcription factor. NF-KB NF-kB is involved in a gene transcription program underling inflammation. When the system is active, there is the activation of a cellular response mediating the production of pro-inflammatory cytokines, etc. This transcription factor is activated by extracellular proinflammatory stimuli, like TNFα and IL-1β which activate their receptors that transduces the signal starting the protein kinase system called IKK α, β and γ. When IKK is active, it will phosphorylate IKBα (inhibitor of NF-kB) causing the release of two other subunits called p65 and p50 (when IKBα is associated to the two protein it stays in the cytosol and can’t migrate to the nucleus) and its degradation. NF-kb (p65+p50) can now go the nucleus and activate the transcription of cytokines, adhesion molecules, tissue factors, MMP9 and acute phase proteins genes. NF-kB can be deacetylated by histone deacetylases and becomes more active. 20 NUCLEAR RECEPTORS AND REGULATION OF METABOLISM At the center of transcription regulation there are the transcriptional activators, which are proteins containing several key functional domains:  DBD: DNA binding domain; it recognizes and binds a specific DNA sequence, which is the regulatory sequence in a promoter or enhancer region;  SSD: signal sensing domain; it can sense an extracellular signal;  TAD: transcription activation domain; it’s the center portion of the transcriptional activator responsible of activating the transcription of target genes; A particular family of transcriptional activators are nuclear receptors, which are defined as ligand-dependent transcription factors. What does it mean? The activity of these factors can be modulated by small molecule that binds to them. They can be classified in three main groups according to certain features:  Endocrine receptors have high affinity for hormonal lipids (KD is in the order of nanomolar: at nanomolar concertation the hormone can bind and activate the receptor). Among this group we have: o ERα-β: estrogen receptor; o RAR α, β, γ: retinoic acid receptor o PR: progesterone receptor; o TR α, β: thyroid receptor o AR: androgen receptor; o VDR: vitamin D receptor o GR: glucocorticoids receptor; o MR: mineralocorticoid receptor;  Adopted orphan receptors: originally, when these receptors where identified, the ligand was not known, therefore they were named “orphan receptors”. Subsequent studies, led to the discovery of ligands of some of these receptors and so they were called “adopted”. Among these we have: o RXR: a receptor for another form of retinoic acid; o PPAR α, β, γ/δ: the physiological ligands are long chain unsaturated fatty acid; o LX*R α, β (Liver X Receptor): the reason of the name was because, originally, it was identified in the liver but it turned out that other tissues and cell types express it (subtype β is ubiquitously expressed), the physiological ligands are oxidized derivatives of cholesterol, the so-called oxysterols; o FXR (Farnesoid X Receptor): it was named after what it was thought was the ligand farnesoid molecules, which are intermediate in the mevalonate pathway of cholesterol (these molecules can bind the receptor at non-physiological concentrations); subsequent studies discovered that the physiological ligand are bile acids; o PXR and CAR: nuclear receptors involved in the activation and regulation of genes involved in xenobiotic and drug metabolism; *The X refers to the orphan concept. 21   Orphan receptor: no ligand is known for this group; except for a couple of them like LRH-1 (Liver Receptor Homologue-1): it was found that maybe some phospholipid can bind and activate it. Most of them are constitutively active: they are active event without the ligand; LOCALIZATION Type I nuclear receptors, when not activated, are not in the nucleus but they are localized in the cytosol: in order to migrate to the nucleus, the ligand must bind. When not associated to the ligand, the receptor is associated to a protein complex containing heat shock proteins (hsp), this complex sequestrates the receptor is the cytosol and consequently cannot properly regulate gene transcription. When the ligand binds the receptor, something happens and the receptor dissociates from the complex and only at this point can move to the nucleus. Nuclear receptors behaving this way are: estrogen receptors, progesterone receptors, androgen receptors, glucocorticoid receptors, mineralocorticoid receptors. These receptors bind to the DNA as homodimers: two subunits of the same protein. In particular, when the hormone binds the hsp complex dissociates unmasking the nuclear localization sequence on the protein and therefore the nuclear receptor can be transported to the nucleus by transporter proteins. In the nucleus, the receptor associates with transcriptional co-activators which participate in the activation of PIC containing RNA polymerase and therefore, gene transcription is promoted. Type II nuclear receptors are already in the nucleus and attached to the consensus sequence even in the absence of the ligand but they are not active. Upon binding of the ligand, the nuclear receptor becomes activated and modulates the transcription of target genes. Nuclear receptors behaving this way are all the other receptors that we have discussed earlier. The receptors usually form heterodimers with RXR (usual partner). For example, thyroid receptor heterodimerizes with RXR and can bind the DNA. Exceptions are: LRH-1 (which bind as a monomer), HNF-4 (which binds as homodimer). Investigators in the field of nuclear receptors used to say that RXR is a promiscuous partner. The partners of RXR can be permissive or not permissive and therefore we can have an active partner or a submissive partner. Examples:  Thyroid receptor heterodimerizes with RXR. When the heterodimer forms, it can be activated only by the ligand of TR, not by the one of RXR. Therefore, TR is not a permissive partner and so RXR is a passive partner;  PPARα heterodimerizes with RXR. Within this heterodimer, both PPAR and RXR can participate in the activation with their own ligands. Therefore, PPARα is a permissive partner and so RXR is an active partner; In particular, in the absence of the ligand, the heterodimer is already sitting on the DNA recognizing specific sequences called responsive elements. Under this condition, the receptor is associated to proteins called co- repressors which keep the transcription of target genes off. When the hormone binds the nuclear receptor, 22 something happens whereby the co-repressor dissociates and the co-activator is recruited and contributes to the activation of PIC containing RNA polymerase; as a consequence, transcription of specific sets of mRNAs are transcribed and will be translated in specific sets of proteins giving the final cellular response. MODULAR ORGANIZATION Nuclear receptors are characterized by:  DNA binding domain, which is important for the dimerization process and contains a structural motive called zinc-finger which is formed by two loops where there are cystine residues that coordinate zinc ions. This motive is important because it determines the specificity of the DNA sequence: depending of slight differences in the structure, the nuclear receptor can bind to a given DNA sequence or another.  Toward the N-terminal portion there is the A/B region containing a functional domain called AF-1 (Activation Function-1) which is an activation domain that is ligand-independent: the transcriptional activity mediated by this region of the nuclear receptor does not require a ligand. Usually, it activates the transcription of target genes by post-transcriptional modifications such as phosphorylation of a particular residues, acetylation and metalation;  Ligand binding domain, this portion is also involved in the dimerization and it’s where a given ligand binds. Within this region we have another activation function called AF-2 and this is ligand-dependent which means that it requires a ligand in order to be active and activate gene transcription; FUNCTIONS REGULATED BY NUCLEAR RECEPTORS High affinity ligands:  Sex hormones: reproduction and sex related characteristics;  Glucocorticoids: glucose and lipid homeostasis;  Mineralocorticoids: electrolyte homeostasis;  Thyroid hormones development, metabolism. Activate the thermogenesis in brown adipose tissue;  Retinoids: morphogenesis, development, differentiation, metabolism; Low affinity ligands:  Fatty acids and prostanoids: fatty acid β-oxidation in the liver (PPARα), in skeletal muscle and in adipose tissue (PPARβ/δ), lipid accumulation in adipose tissue and adipocyte differentiation (PPARγ), pesticides, ftalates and hypolipidemic drugs;  Oxysterols: glucose and lipid metabolism;  Bile acids: glucose and lipid metabolism; 23 3D-STRUCTURES Why can we switch the on or off depending on the presence or absence of the ligand? The answer comes from the crystal structure of some nuclear receptors. The first nuclear receptor structure was solved in the 90s, the researchers crystallized the ligand binding domain of RARγ and RXRα. The first one was crystallized in the presence of the ligand (holoreceptor), in the second case the structure was solved in the absence of the ligand (aporeceptor). N.B. Holoprotein: associated with another molecule, which can be a ligand; apoprotein: part of the complex including only the specific protein without the ligand. The striking difference determined by the presence of the ligand is the conformation of helix12:  In the presence of the ligand, H12 closes the ligand binding pocket like a lid;  In the absence of the ligand, H12 stays outside the ligand binding pocket; As a matter of fact, H12 has been considered the functional portion of the nuclear receptor corresponding to AF-2. Depending on the orientation of helix12, we can have active, or non-active, nuclear receptor. In case of type I nuclear receptors, the presence of the ligand changes the structure of the receptor in such a way that it will not be capable of interacting with the hsp complex and consequently there is the dissociation and the migration to the nucleus. The conformational switch is important in the activation because co-activators will be able to bind and interact physically only if there is an optimal conformation of the nuclear receptor: the two surfaces become sort of complementary with each other. Vice versa, in the absence of the ligand, the surface of the receptor will be optimal for interact with co-repressors. There is one nuclear receptor which called small heterodimer partner (SHP) that is unique because it doesn’t have a DNA binding domain, therefore it cannot bind the nucleic acid directly therefore it heterodimerizes with other nuclear receptors/transcription factors and acts as a co-repressor. For example, SHP can heterodimerizes with HNF-4 and LRH-1 and represses the transcription mediated by these receptors. The structure of nuclear receptors must be different because they can bind different ligands. Chemically speaking the ligand forms reversible bonds (hydrogen bonds, electrostatic interaction, Van der Vaals interactions) with the ligand binding pockets. In the primary sequence of the protein, we may have different ammino acid residues which can mediate interactions with a given ligand. This is why each nuclear receptor is specific to its own physiological ligand. When the receptor is associated to an agonist, its helixe12 adopts the optimal conformation in the space causing the optimal interaction with a co-activator protein. Vice versa, in the presence of an antagonist (molecule which prevents the effects of the agonist), H12 adopts a different orientation in the space and this conformation is not optimal for the interaction with a co-activator but it’s more optimal for the interaction for 24 a co-repressor (especially for type II receptors). What dictates these two different conditions? The agonist and the antagonist are two chemically distinct molecules, this means that they set a different network of bonds with the ammino acids located in the ligand binding pocket of the receptor. So, depending of the network of chemical bonds we set, we can force the nuclear receptor either in the conformation optimal for the activation of gene transcription, or the one not optimal at all. These are two opposite conditions. We can design a chemical ligand which binds the ligand binding pocket in such a way that it will confer a structure to the nuclear receptor which can be intermediate. This ligand is defined as a partial agonist (or antagonist): a molecule that acts like an agonist but cannot fully activate the receptor and so the activation is much weaker. From a pharmaceutical point of view, this is interesting because not always the full activation of a nuclear receptor will give only beneficial effects, there can be also side effects; therefore, we use partial agonist in order to provide the beneficial effects and avoid the unwanted effects that we would have if we fully activate the receptor. These molecules are called selective modulators of nuclear receptors. Since the receptor is partially activated, H12 will be in an orientation that allows the recruitment of some co- activators and also of some co-repressor. For one nuclear receptor there is not just one co-activator and one co- repressor, but there are many different activators and repressors. Depending on which co-activator and which co-repressor the receptor recruits, we may have slightly but biologically important differences in the pharmacological response. In different tissues we can have differential effects of the same ligand depending on which activators and repressors are recruited. HOW CAN WE STUDY NUCLEAR RECEPTORS? While studying a nuclear receptor we want to create a new synthetic ligand which, to do so we do a screening of thousands of chemical compounds in order to select the one that binds the receptor the most. A typical screening procedure is called cell-based assay, in which we test the capacity of each single molecule in the chemical library to activate the transcription mediated by the nuclear receptor of interest. We need a reporter system that contains the reporter gene which is luciferase that is under the control of a DNA consensus sequence called GAL4 (a yeast transcription factor). In addition to the reporter gene, we have a fusion protein that consist in the GAL4 DNA binding domain and the ligand biding domain of the nuclear binding receptor of interest (chimeric protein). In the cellular system, only the chimeric protein is able to bind and promote the transcription of luciferase. Once we insert the fusion protein and the plasmid of the reporter gene in the cellular system, we add to different wells different ligands. Once the incubation period finishes, we select only the plates where we can find a positive result given by luciferase. We can have false positive because there could be metabolic changes of the molecule that we inserted  the metabolite of the original molecule binds the nuclear receptor). 25 We use the GAL4 system because there is no interference with any endogenously expressed nuclear receptor; only the Gal4 chimera can bind the consensus sequence. There is also a greater activation with agonists. PPAR S (PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS) Peroxisome Proliferator Activated Receptors are interesting because the three different subtypes are expressed in different tissues and therefore, they regulate different processes. Subunit Expression Function Liver Catabolism of triglycerides and fatty acid α Heart Reducing inflammation Smooth and skeletal muscle cells Reducing inflammation Adipocyte differentiation γ Adipose tissue Glucose homeostasis Insulin sensitivity Wound healing Reducing inflammation β/δ Ubiquitous Triglycerides and fatty acid catabolism in skeletal muscle and adipose tissue The physiological ligands are long chain unsaturated fatty acid, there are also molecule on the market that can activate some of these receptors:  Fibrate, in particular fenofibrate which largely used to prevent side effects of hyperlipidemia and possibly cardiovascular diseases. It reduces levels of triglycerides by promoting fatty acid catabolism, mainly in the liver and also in skeletal muscle;  Glitazones: type II diabetic agents, they were used but nowadays not so much due to some side effects; Researchers used the Gal4-based assay to study new synthetic PPAR ligands, they tested the two enantiomers of a given ligand: It resulted that the S-enantiomer is a partial agonist because at high concentrations the final effect is half the effect, we would get with rosiglitazone, a known full agonist  we never reach a full activation. R-enantiomer is a full agonist but it has lower affinity than rosiglitazone because we need more concentration to have the same result. Both enantiomers are less potent than rosiglitazone. Are these compounds PPARγ ligands? Do these compounds bind and affect the receptor activity? The cell-based assay cannot tell us whether the molecule we’re administering to cell directly binds the nuclear receptor; this assay tells us that the molecule somehow results in the activation of the transcription mediate by the nuclear receptor, but we don’t know if it’s a metabolite or if it’s the actual ligand. 26 How can we make sure that a given molecule is the actual ligand? There are couple of tests that can help us answering this question, one of them is the scintillation proximity assay (SPA) which is a cell-free assay (chemical assay). Reagents:  Derivative of the nuclear receptor containing the ligand binding domain which contains the so-called tag, that can be either histidine tag or GST tag (Glutathione S Transferase), in this way we generate a chimeric protein using recombinant DNA techniques. In order to have the protein we need to express it in E.coli and to purify it or, more simply, we can buy purified receptors;  Testing molecules and reference molecules (e.g., rosiglitazone). We have to radiolabel the testing molecule; we can also radiolabel the reference molecule in order to do a competitive assay, by adding increasing concentration of cold and not labeled testing compounds we can create the competition curve;  Beads covered with molecules which can recognize the tag on the chimeric receptor. For example, for recognizing the histidine tag we use nickel. Inside these beads there is a scintillation liquid which can emit light if the radioligand in close proximity to the bead. In order to emit light, the bead needs to be in close proximity to the radioligand, this happens only if the radioligand is associated to the nuclear receptor. In the scintillation counter we then measure radioactivity; Another assay is called coactivator recruitment assay that exploits the principle of FRET (Fluorescence Resonance Energy Transfer). This is also a cell-free assay, we can use the same reagents mentioned before: ligand binding domain of the nuclear receptor that carries a tag and testing molecules. This assay is based on the interaction between the nuclear receptor and a co-regulator protein, either a co-activator or co-repressor, therefore another reagent to put in the microplate is a small peptide containing the region of the co-regulator that physically interacts with the receptor and this small peptide is labelled with biotin (biotinylated). The last reagent is streptavidin (interacts with biotin) which is labeled with a fluorescence dye called SA/XL665 or allophycocyanin that emits light at650nm when excited with light at 620nm. The light at 620nm comes from another reagent which is an antibody that recognizes the tag protein; the antibody is labeled with a lanthanide element, in this case is europium (Eu) which emits light at 620nm if we excite it with light in the UV range (precisely at 337nm). Under what condition can we measure light at 665nm? In order to have light emission at 665nm, the streptavidin labeled with allophycocyanin must be close enough to europium in order to be able to capture the light at 620nm 27 and therefore the fluorescence molecule will be excited and emitting light at 665nm. Actually, what we measure is the ratio between 665nm and 620nm. The ligand binds the receptor and elicitates the conformational change that allows the interaction between the nuclear receptor and the co-regulator (this brings in close proximity europium and allophycocyanin). This assay is performed in a lot of plates and therefore we can screen many compounds at the same time. This process is called FRET because energy is transferred from excited europium to allophycocyanin. We can also test whether a given ligand can preferentially recruit different co-regulators. In C they fixed the concentration of the nuclear receptor chimeric construct and of the co-activator peptide and put increasing concentration of the reference ligand (rosiglitazone) and of the R- and S-enantiomer. We obtain similar curves as the ones seen before: the R-enantiomer is a full agonist, while the S-enantiomer is a partial agonist. At the same time, we can see that the two chemical ligands have a higher IC50 compared to rosiglitazone: we need higher concentration in order to elicit the interaction between nuclear receptor and co- activator. Based on the result obtained in C, we would assume that the affinity of R-1 and S-1 would be different, lower compared with rosiglitazone. But the FRET assay is not actually a direct measurement of the affinity of the ligand, the reason is because it depends on what co-activator we use. It might be that rosiglitazone preferentially recruits SRS1 (co-activator used in the assay), whereas, for example, R-1 has a lower preference. It might be that if we screen other co-activator peptides, we may have similar curves when we compare rosiglitazone to R-1. In other word the FRET assay does not give us a measurement of the affinity for a given ligand but it’s very important because it can provide important information about the preferential recruitment of different co-activators or co-repressors. If we want to get the right affinity value for the tested molecule, we need to use the scintillation proximity assay. As a matter of fact, when we perform the assay (E) we notice that the curve of R-enantiomer and rosiglitazone are overlapping meaning that the two ligands have similar, or comparable, affinity; while the S-enantiomer’s curve is shifted and has a lower affinity. In this case they used a competitive SPA assay: they used fixed concentration of rosiglitazone and then they added increasing concentrations of the cold enantiomers. They did the competitive assay because it would have been too expensive to label the two enantiomers, instead the bought the rosiglitazone already labeled. PPARγ, until 15 years ago, a very exciting and promising target of type II diabetes because is highly expressed in adipose tissue where it regulates the expression of GLUT4 which is important for internalizing glucose in response of insulin and therefore its activation is considered a good way to increase insulin sensitivity. But unfortunately, in 2005 and 2007 there were some reports showing some troubles with rosiglitazone (PPARγ based drug): it came up that this drug had increased risk of developing some side effects due to myocardial infraction and death for cardiovascular causes. It turned out that rosiglitazone, and also pioglitazone, had some side effects among which there are:  Excessive adipogenesis, this makes sense because PPARγ is also a regulator of adipogenesis;  Excessive incidents of bone fractures; 28   Effects at the cardiovascular level due the fact that rosiglitazone increases the expression of sodium transporter in the kidney causing excessive reabsorption of sodium and water and therefore blood volume increases which results in cardiac problems; Based on these observation, FDA and other regulatory agencies decided to ask companies developing new molecules targeting PPARγ and PPAR in general, more assays performed at the pre-clinical level to test toxicity, side effects and so on. This killed pharmaceutical researches on this target, because all these tests performed on animal models are very expensive and therefore companies decided that pursuing this type of target wasn’t worth it. A researcher observed that when you treat mice with a high fat diet, which is a way to make them obese and insulin resistant, the nuclear receptor PPARγ becomes phosphorylated on Ser273 and this phosphorylation dysregulate the activity of this receptor causing the unproper activation of a subset of genes. In other words, obesity and type II diabetes develop because PPARγ gets phosphorylated and this makes it not properly functional and all the transcription programs of adipocytes are dysregulated as a consequence. Therefore, the idea was to avoid the phosphorylation on the serine residue. In the image below (a) we can see a Western blot showing treatment with high fat diet at 3, 7 and 13 weeks and the assay detects phosphorylated PPARγ on Ser273; we can see that with high fat diet (HFD) at 13 weeks the signal is much stronger than the mice treated with chow diet (normal diet). In particular, this was evident in epididymal fat and also in inguinal fat (b): in both we can see the increase in phosphorylation. The researcher observed that when these mice were treated with rosiglitazone, could prevent the phosphorylation of the serine residue. The problem is that we know that rosiglitazone can cause some unwanted effects. To avoid the side effects, they came up with a screening on SR1664 and SR1824 (which are non-agonist ligands) where the performed a GAL4 based assay in the presence on increasing concentrations of rosiglitazone. In this experiment, even at higher concentration of the two molecules did not activate PPARγ, in particular they don’t bind it but they prevent the phosphorylation of Ser273. In order to understand if these molecules could have a future in diabetes therapy, they performed other functional analysis in vitro. An overactivation of PPARγ, leads to increased incident of bone fractures and the reason is because PPARγ controls the differentiation program of adipocytes starting from stem cells, but the point is that adipocytes precursors are also precursors of osteoblasts which are cells important for proper bone metabolism. PPARγ acting on these progenitor cells, pushes the differentiation program towards adipocyte at the expense of osteoblasts differentiation. Based on this knowledge they performed some experiments like the staining with alizarin red (c, used to check the possible formation of osteoblast) in which they saw that rosiglitazone decreases the staining whereases the two ligands not activating the receptor but preventing the phosphorylation did not reduce the staining. At the same time, they checked adipogenic potential (a), to do so 29 you need to differentiate adipocytes in culture in the presence of the experimental ligand or the reference molecule and then you stain these cells with Oil Red O which is a red oil that binds to lipids (more lipids in the adipocytes the stronger is the red color). To get some molecular insights on the effects of these molecules, they measured the level of mRNA of typical targets of PPARγ (b), like: aP2, C/EBPα, GLUT4, etc. In the presence of rosiglitazone, the level of these mRNAs, as expected, increases; instead, in the presence of the non-agonist ligands, the mRNAs levels don’t increase. To understand what happens when they treat mice with these molecules, they treated them with different dosages of SR1664 while they were on a high fat diet (HFD). They first detected the phosphorylation of PPARγ (a) and we see that obese mice have a higher level of phosphorylation, whereas at all three dosages the phosphorylation is reduces and therefore prevented. Glucose concentration in the blood was not significantly reduced (b1), although there was a tendency to reduce fasting glycemia. But the important parameter was the concentration of insulin in the blood (b2) which was reduce significantly at 10 and 20 mg/kg; this data is important because it reflects the insulin sensitivity. They also show results with HOMA-IR index (b3) which and index of insulins resistance, the higher is this value, the higher is the insulin resistance in a given sample, and they saw that with the three dosages insulin resistance is decreased. The golden assay for testing insulin resistance is the euglycemic insulin clamp: you give a shot of insulin to the experimental model and then you measure the rate of glucose that we have to infuse in order to keep glycemia constant, if we don’t do so the glycemia decreases because insulin reduces it. The higher is the rate of glucose infusion, the higher is the sensitivity of the animal that we are testing  if we need to infuse more glucose it means that insulin is more effective and consequently the model is more insulin sensitive. They also measured the capacity of taking up glucose in adipocytes (c), the experimental molecule was able to increase the glucose uptake and the suppression of hepatic glucose production. 30 To summarize, this molecule can help to treat the diabetic phenotype and at the same time can prevent the possible side effects typically associated with PPARγ agonists like rosiglitazone. This is a potential way to develop new molecules. The take home message is: when we discover a potential pharmaceutical target, we don’t need to pursue it with a molecule which has high affinity and can activate it (this is a bad idea because higher is the potency, the higher is the affinity of the compound to the target, consequently it means that we don’t have a wide therapeutical window), but it’s wiser to design molecules that have moderate effects on the target and sometimes it is enough to obtain the beneficial effects and avoid potential side effects due to excessive activation of a given receptor. LT175 is a dual PPARα/γ ligand. The double activation of both PPARα and PPARγ, ideally, could give more beneficial effects than the activation of just one of the two: the activation will be the liver and in the adipose tissue, in this way we attack parameters that are altered in diabetes in two tissues at the same time with one molecule. They tested whether LT175 was a full or partial agonist of PPARγ, to do so they performed the GAL4 based assay with increasing concentrations of rosiglitazone and LT175. From the data shown in the graph, we can say that LT175 is a partial agonist and it’s less potent than rosiglitazone since we need higher concentrations in order to reach the maximum effect. 3T3-L1 are murine fibroblast that can be differentiated to adipocytes in culture. After differentiating these cells for seven days in presence of insulin and our ligand they measured the expression of genes target of PPARγ and they performed lipid staining with Oil Red O. They observe that with LT175 less lipids accumulation in adipocytes. This ligand is less adipogenic as rosiglitazone. Why? Because it has been observed that LT175 is oriented in the ligand binding pocket of the receptor in a different manner compared to farglitazar, in particular LT175 goes deeper. Also, the orientation of Phe282 is very different. They performed also a co-regulator recruitment assay of PPARγ in the presence of the fixed concentration of rosiglitazone and of the tested ligand. In this case, they didn’t use increasing concentration because they wanted to test the preferential recruitment of different co- activator or co-repressor peptides elicited by either the reference compound or the experimental molecule. There are some striking differences:  Rosiglitazone recruits PGC-1 and CPB (co-activators), while doesn’t elicit at all the recruitment if NCOR (co-repressor)  LT175 recruits PGC-1 and NCOR, while CBP is no longer efficiently recruited; Since LT175 can elicit the recruitment of a co-activator and a co-repressor, we can conclude that this molecule is a partial agonist. Depending on the abundance of a given co-regulator in different cell types, this ligand may behave as an agonist or an antagonist: it may limit the full activation of the receptor. Another important point is that CBP is not recruited at all by this ligand. They tested this molecule in vivo: they administer it to diet induced obese (DIO) mice. Before sacrificing the animals, they measured fat content using MRI. In the pictures, we can observe that mice treated with normal diet do not have a lot of visceral adipose tissue, mice treated with high fat diet have a lot of visceral adipose 31 tissue and then mice treated with high fat diet and with LT175 we can see that there is a clear reduction of the fat. When they measured the expression of some PPARγ target genes, they detected that rosiglitazone is capable of increasing the expression of almost all the genes at a much higher levels as compared to the same genes in animals treated with LT175. Therefore, the compound is less adipogenic. They also performed Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT), two commonly used tests to determine insulin sensitivity and diabetes. Hight fat diet (HF) mice have higher insulin resistance compared to mice with normal diet. Mice treated with LT175 have an even better curve of glucose tolerance while testing glucose and insulin tolerance test. Conclusions:  LT175 is a full PPARα agonist and behaves as a partial PPARγ agonist. o PPARα is expressed mainly in the liver and it’s involved in fatty acids β-oxidation (in general triglycerides catabolism) meaning that we can promote induction of fatty acid accumulation. In other words, we can burn more fat. This nuclear receptor is also expressed in skeletal muscle which can help to burn more fatty acids. This would be an advantage in the context of obesity/overweight because it would decrease the amount of fat/excess of adipose tissue; o PPARγ is mainly expressed in adipose tissue, its partial activation is beneficial because increases GLUT4 expression, in this way it increases insulin sensitivity lowering glycemia level which is helpful in type II diabetes. Adiponectin is another very important target of PPARγ and the molecule is called also “adipokine” which has antidiabetic-insulin sensitizing effect. Adipocytes secrete adiponectin which then has systemic positive beneficial effects, one of them is on the skeletal muscle: it activates the transcription of GLUT4. Is important to target skeletal muscle in order to decrease glycemia because muscle tissue is widespread in our body so it’s the most responsible for clearing the excess of glucose;  LT175 induces a unique conformational change in the PPARγ ligand binding pocket allowing the access to the diphenyl pocket;  LT175 induces PPARγ-dependent adipogenesis with low lipid accumulation;  in vivo LT175 strongly improves lipid and glucose homeostasis and reduces fat content;  It has been shown that recruitment of CBP leads to more lipid accumulation in adipocytes, therefore the possibility of selectively recruit some co-regulators may be the key of these special features of this molecule that potentially could have more beneficial effects that rosiglitazone; 32 EPIGENETICS  Epigenetics refers to a set of processes that regulate gene expression without modifying DNA sequence;  The best known and most frequently studied epigenetic processes involve DNA methylation and histone modifications. Specific epigenomic signatures are associated with specific cell types; as mammalian organisms, we are very complex containing different organs and tissues and each cell type is specific, even within the same tissue we have a variety of subpopulation of cells that are slightly different with each other and these differences may be because of different epigenomic signatures;  Epigenetic programming of gene expression affords the genome its remarkable plasticity, allowing for one compliment of DNA or a single genome to give rise to the multiple phenotypes associated with diverse array of unique cells and tissues found in our bodies:  Each cell in a single human has the same compliment of gen