Insulin Definitivo

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    Maria Teresa Aranda, Arnzazu Arrieta, Brbara

    Ballester, Sergio Ballesteros, Marina Belda, Isis Bonilla,Maria Caams, Macarena Campos


    Jose Enrique OConnor

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    Insulin is a hormone that regulates carbohydrate and fat metabolism in the body. It can

    be found in liver, all muscles, and fat tissue in order to take up glucose from the blood and store

    it as glycogen in the liver and muscles. It is produced in the islets of Langerhans in the pancreas.

    Insulin impedes the use of fat as an energy source inhibiting the release ofglucagon.Its

    also a central metabolic control mechanism of other body systems (such as amino acid uptake

    by body cells).

    A failure in the control of insulin levels results in diabetes mellitus. As a consequence,

    insulin is medically used to treat some forms of diabetes mellitus. There are two types of

    diabetes depending on the production or not of insulin: type 1 and type 2. In type 1 the hormone

    is no longer produced internally while in type 2 insulin is produced but in a deficiency amount.

    The name comes from the Latin insula for "island". Insulin's structure varies slightly

    between species of animals, indeed porcine insulin is especially close to the human version.

    Thanks to several discoveries about insulin, their authors have won the Nobel Prize

    during the 20 century. For instance, insulin was the first protein to have its amino acid sequence

    sequenced, in 1955 by Fred Sanger (Sanger 1988), earning him a Nobel prize in 1958. It was

    also the first peptide hormone, circulating in minute amounts, to be measured by

    radioimmunoassay (Berson and Yallow 1961), earning Yalow a Nobel Prize in 1977. The

    pathway behind the biosynthesis of insulin in pancreatic beta cells, specifically as a proinsulin

    precursor, was determined by Don Steiner in 1967 (Steiner and James 1992). The three-

    dimensional structure of insulin was ultimately solved by Dorothy Crowfoot Hodgkin and

    colleagues in 1969, using X-ray crystallographic methods (Adams et al. 1969). It was also the

    first protein to be synthesized in microorganisms by recombinant DNA technology in the late

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    1.The Structure of InsulinInsulin is a pedtidic hormone composed of two polypeptide chains: chain A has 21 amino

    acids and chain B has 30 amino acids (in humans). That sums a total of 51 amino acids linked

    by two disulfide bridges (residues A7 to B7, and A20 to B19). These are covalent bonds that

    tether both chains. Chain A also contains an internal disulfide bridge (between the residues A6

    to A11).

    This is an endocrine hormone, which means that it is secreted into the

    blood stream through which it travels to affect distant organs. It has a

    compact three-dimensional structure (insulin monomer is essentially the

    same in solution and in solid phase) consisting of three short helices and

    three invariant (conserve) disulfide bridges. This basic fold (three-dimensional) is present in all members of the insulin peptide family,

    despite divergent sequences.

    Due to the fact that insulin is a protein, it has four structural levels.

    Insulin and insulin preparations during storage and use can be affected by a variety of chemical

    changes of the primary structure (yielding insulin derivatives) and physical modifications of the

    secondary to quaternary structures (resulting in "denaturation," aggregation, and precipitation).

    Insuline mainly exists in two main quaternary conformations that can adopt a

    therapeutically-significant "R" or "T" state (both are hexamers, therefore they are used in

    preparations for therapy). These differ in the extent of helix in the B chain which is governed bythe presence of phenol or its derivatives. In acid and neutral solutions (in micromolar

    concentrations relevant for pharmaceutical formulation) the insulin monomer assembles to

    dimers while in neutral pH (in the presence of Zinc ions) it further associates into hexamers.

    When the A chain has an amino-terminal helix (A1-A8) linked to an antiparallel carboxy-

    terminal helix (A12-A20) and the B chain has a central helix (B8-B19), flanked by extended

    amino- and carboxy-terminal strands this arrangement is called the "T" conformation. On the

    other hand "R" conformation exists where the B chain helix extends from the N-terminus (B1-

    B19, versus B8-B19). In the 4-Zn hexamer, three of the monomers are in the T form and three

    are in the R form (R3T3), as a result of a high chloride concentration. An R6 form exists in

    phenol-containing crystals and in solution

    An allosteric equilibrium controls the T-R transition, which plays an important role in the

    formulation of therapeutic insulin where chloride is used as an isotonic agent and phenol is used

    as an antimicrobial agent.

    Despite the fact that insulin has different conformations, the biologically active one is the

    monomeric conformation.

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    Stability of the structure: The insulin hexamer forms a relatively stable unit but some

    flexibility remains within the individual molecules. The intrinsic flexibility at the ends of the B

    chain plays an important role in governing the physical and chemical stability of insulin. One of

    the things that contribute to protein stability is a cluster of hydrophobic residues that form the

    core of the small protein.

    2.Biosynthesis of insulinInsulin is synthesized in the pancreas. This organ can act as an exocrine or an endocrine

    system. The endocrine function is executed by a group of small structures called islet of

    Langerhans (1% of the pancreatic mass) which are composed of different cells that produce

    hormones. There are 4 types: (A), (B), (D) and Pp (F).

    The cells are localized in the middle of the islet. They are activated when the organism has

    metabolism fuel like glucose, aminoacids These nutrients induce the release of someproducts: insulin, c-peptide, amylin and sometimes proinsulin. Although insulin is the main

    product, c-peptide is released in the same molar amount as insulin. Amylin is a small peptide

    that appears in diabetis type II and proinsulin appears when it hasnt be removed (incorrect


    The cells are situated in the periphery of the islet. They become active in stress situation,

    that means when the organism has a deficiency of nutrients (glucose, aminoacids). They

    release glucagon to the blood stream in order to mobilize energy and release glucose from the


    The cells have a suppressor function. They inhibit the release of products by the and cells by a hormone called somatostatin (GHIH: growth hormone-inhibition hormone). They are

    located overall within the islet.

    This inhibition by the cells is due to their innervation. The artery enters directly in the

    middle of the cell irrigating first the cells. Then the capillaries spread towards the periphery

    and the venules take the stream into the veins. This direccion allows that whatever the cells

    produce, passes through the and cells so they can act in response. This is called a paracrine


    To explain in more detail how the

    insulin is synthesized, we must followthe route of synthesis inside the cell.

    Inside the nucleus, the gene that

    codifies for insulin is in the

    chromosome 11. Once this chromosome

    is converted into chromatin and opened

    for the transcription, mRNA can be

    formed. Then it will go to the

    cytoplasm, and several ribosomes will

    bind to this chain in order to execute the

    translation. When a peptide chain startsto form, ribosomes go to the

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    endoplasmic reticulum (rough endoplasmic reticulum) and the different chains of aminoacids

    enter inside the lumen.

    Its inside the lumen where the precursor of the insulin, called preproinsulin, is formed. It

    has an initial segment (signal sequence), an amino-terminal B-chain, a connector segment (C-

    chain) and a carboxy-terminal A-chain. At this place preproinsuline is exposed to severalspecific endopeptidades (proteolytic enzymes) that break the signal sequence, therefore the pre

    compartment is lost.

    This molecule now called proinsuline has three disulphide bonds: two between the B and A

    chain and one within the A-chain. At this moment the molecule is folded in a vesicle and

    transported to the golgi apparatus. There it is cut again by the endopeptidases, but this time the

    C-chain (which was between the B and the A chains) is deleted. Now the mature insulin is

    formed. It is stored in vesicles (in the cytoplasm) ready to be released. Finally its in this

    secretory granules where the mature insulin and the C-peptides are and this is the reason why

    they are in the same amount. Zinc and proinsulin can also be found when the maturation hasnt

    been completed.

    When the beta cell is appropriately stimulated, insulin is secreted from the cell by exocytosis

    and diffuses into islet capillary blood. C peptide is also secreted into blood, but has no known

    biological activity.

    As most of the processes in the organism, this endogenous production of insulin is regulated

    in several steps along the synthesis pathway, for example: at transcription from the insulin gene,

    in mRNA stability, at the mRNA translation, in the posttranslational modifications, etc.

    3. Pharmacological types of insulinBovine or porcine insulins have nearly disappeared from the market, displaced by human

    insulin obtained by genetic engineering. All current preparations have been submitted to a

    process of ultrapurification to eliminate any foreign proteins.

    The different types of insulin are categorized according to how long it takes to begin

    working (onset), when it's working the hardest (peak) and how long it lasts (duration). The types

    now available include rapid-, short-, intermediate-, and long-acting insulin.

    Doctors will work in developing an insulin regime that works specifically for each person,his health and his lifestyle. People should never make changes in their dosage without first

    consulting with their doctor. These insulin regimes consist in prepared mixtures of fast with

    intermediate insulin which are nowadays available on the market, with the purpose of

    obtaining a quick start and long duration medication. These prepared mixtures are called

    biphasic insulins and are basically Humulin, Humalog, Mixtard and Novomix. Usually people

    who take insulin use a combination of a rapid- or short-acting and intermediate- or long-acting

    insulin. This helps keeping blood sugar levels within a range that is safe for the body throughout

    the day.
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    Nowadays the main types of insulin are:


    Generic Name Brand Name Action Doing schedule

    Insulin aspart Novorapid Onset: 10-15 minutes.

    Peak: 60-90 minutes.

    Duration: 3-5 hours.

    Usually taken right

    before eating, or to

    lower high blood


    Insulin glulisine Apidra

    Insulin lispro Humalog

    Insulin regular Humulin-R Onset: 30 minutes.

    Peak: 2-3 hours.

    Duration: 6.5 hours.

    Taken about 30 minutes

    before eating, or to

    lower high blood



    Generic Name Brand Name Action Doing schedule

    Insulin NPH Humulin-N Onset: 1-3 hours.

    Peak: 5-8 hours.

    Duration: up to 18


    Often taken at

    bedtime, or twice a

    day (morning and



    Generic Name Brand Name Action Doing schedule

    Insulin detemir Levemir Onset: 90 minutes.

    Peak: none.

    Duration: up to 24

    hours (Lantus 24 hours,

    Levemir 12-24 hours).

    Usually taken once or

    twice a day.Insulin glargine Lantus

    How insulin is taken

    The usual way of insulin administration is subcutaneous. The most common method

    of administration is using special syringes graduated in units of insulin (IU). Injector devices in

    the form of pen injector have reached acceptance, because they facilitate regimes of multiple

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    injections per day. There are also prefilled syringes capable of dispensing with precision in

    increments of 2 IU and useful for many applications by changing the needle. Infusion pumps

    administrate continuously a basal insulin dose, supplemented by extra doses before meals. Some

    insulin can be given through a vein but only in a hospital. Injectable insulin is packaged in small

    glass vials (bottles) and cartridges that hold more than one dose and are sealed with rubber lids.

    The cartridges are used in pen-shaped devices called insulin pens.

    Research is on going to develop not only new forms of insulin but also insulin that can

    be taken in other ways, such as by mouth. These are the different ways of injecting insulin

    nowadays: pen injector, jet injector or external pump.

    Dosing guideline

    The spread of the idea that strict glycemic control can prevent long term complications

    of diabetes has created a tendency to use dosing schedules aimed to fit as closely to the

    administration of insulin to the diurnal variations of glycemia. This involves multiple daily

    injections regimens and glycemic control by the patient himself. The most commonly used

    guidelines today are:

    - Two doses of a mixture of intermediate and rapid insulin. The popularity of this regime

    explains the spread of biphasic insulin preparations.

    - A daily dose of long-acting insulin and three insulin injections per day fast before the

    main meals. This regimen requires motivated patients, but has the advantage of

    allowing more flexible meal times. Systems have been introduced to minimize its

    drawbacks; one of them is the use of pens-type injectors.

    Side effects

    The major side effect of insulin can be a dangerously low blood sugar level (severe

    hypoglycemia). A very low blood sugar level can develop within 10 to 15 minutes with rapid-

    acting insulins. Insulin can contribute to weight gain, especially in people with type 2 diabetes

    who already are overweight. Other possible side effects of long-term insulin use include the loss

    of fatty tissue (lipodystrophy) where the insulin is injected and, in rare cases, allergic reactionsthat include swelling or edema.

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    Insulin secretion is pulsatile (i.e. increases as needed by bursts) and is regulated by a

    variety of stimulatory and inhibitory factors, most of them related to glucose metabolism andthe effects of cAMP. This explains that Insulin secretion is stimulated by high blood glucose

    levels and reduced when blood glucose is low. This makes sense because insulin is in charge of

    facilitating glucose entry into cells.

    Some neural stimuli (e.g. sight and taste of food) and increased blood concentrations of

    other substances, including amino acids from ingested proteins, fatty acids, acetylcholine

    released from vagus nerve endings (parasympathetic nervous system), and gastrointestinal

    hormones released by enteroendocrine cells of intestinal mucosa and glucose-dependent

    insulinotropic peptide (GIP), also promote insulin secretion.

    These hormons that stimulate insulin secretion are: growth hormone, placental lactogen,

    estrogens, and progestins. These increase its secretion by increasing the preproinsulin mRNA

    and enzymes involved in processing the increased preprohormone. Inhibitory factors include

    somatostatin, norepinephrine (sympathetic stimulation) and others.

    Also, in general, some release takes place with food intake, not just glucose or

    carbohydrate intake, and the -cells are also somewhat influenced by the autonomic nervous


    Our understanding of the mechanisms behind insulin secretion remains somewhat

    fragmentary. Nonetheless, certain features of this process have been clearly and repeatedly


    The - cells in the islets of Langehans release insulin, mainly in response to the

    presence of high levels of glucose in the blood, in two phases. The first phase release is rapidly

    triggered in response to increased blood glucose levels. The second phase is a sustained, slow

    release of newly formed vesicles triggered independently of sugar.

    These cells are quite dependent upon glucose as its substrate for energy metabolism.

    Neither fatty acids nor amino acids can serve as substrates to support high ATP levels which

    play a significant role in this process.

    Also - cells contain several types of channels in their membranes, each of which

    allows a particular type of ion to pass through. These include channels that let K+

    ions pass

    through and others that let Ca2+

    pass through.

    Normally, the K+

    channels are open, leaving the K+

    ions free to pass through. These

    positively charged ions diffuse from inside the cell to outside. This makes the outside of the cell

    positively charged compared with inside. We say that there is a potential difference across the

    membrane. The potential difference across the plasma membrane of a resting -cell - one which

    is not secreting insuline- is about -70 mV.

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    However, when glucose enters into the - cell by facilitated diffusion through the

    glucose transporter GLUT2 the concentration of it within the - cell rise sharply, and this

    increase in glucose levels starts off a chain of events which alters the potential difference of the

    resting - cell. In other words, elevated concentrations of glucose in extracellular fluid leads to

    elevated concentrations of glucose within the beta cell, and as a consequence many processes

    related to the secretion of insulin begin. Once the glucose is inside the - cell it is quicklyphosphorylated by the enzyme glucokinase. Then, this phosphorylated glucose goes into

    glycolysis and the respiratory cycle, where multiple high-energy ATP molecules are produced

    by oxidation.

    As a result, the K+

    channels which are sensitive to the amount of ATP in the cell

    respond to this increase in ATP levels by closing. So now the K+

    ions cannot diffuse out.

    Consequently, the difference in electrical potential on the inside and the outside of the

    memebrane becomes less. It is now only about -30mV, this is called a depolarization of the


    On depolarization, voltage-controlled calcium channels (Ca2+

    ) open and extracellular

    calcium flows into the cells. This increase in calcium levels causes the activation of

    phospholipase C, which cleaves the membrane phospholipid phosphatidyl inositol 4,5-

    biphosphate into inositol 1,4,5-triphosphate and diacylglycerol. Inositol 1,4,5-triphosphate (IP3)

    binds to receptor proteins in the membrane of endoplasmic reticulum (ER). This allows the

    release of Ca2+

    from the ER via IP3-gated channels, and further raises the cell concentration of

    calcium. A significant increase in the amount of calcium affects the behaviour of the vesicles in

    the cells and causes the release of previously synthesized insulin, which has been stored in these

    secretory vesicles, these vesicles are moved towards the plasma membrane, where they fuse

    with the membrane and empty their contents outside the cell. This is the main mechanism of

    insulin release.

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    Clearly, elevated glucose not only simulates insulin secretion, but also transcription of

    the insulin gene and translation of its mRNA.

    Stimulation of insulin release is readily observed in whole animals or people. The

    normal fasting blood glucose concentration in humans and most mammals is 80 to 90 mg per

    100 ml, associated with very low levels of insulin secretion.

    The figure to the right depicts the effects

    on insulin secretion when enough glucose is

    infused to maintain blood levels two to three

    times the fasting level for an hour. Almost

    immediately after the infusion begins, plasma

    insulin levels increase dramatically. This initial

    increase is due to secretion of preformed insulin,

    which is soon significantly depleted. The

    secondary rise in insulin reflects theconsiderable amount of newly synthesized insulin

    that is released immediately.

    The exocrine pancreas has a very important rol in the stimulation of insulin secretion by

    intestinal hormones.

    Two of the many gastrointestinal hormones have significant effects on insulin secretion

    and glucose regulation. These hormones are the glucagon-like peptides (principally glucagon-

    like peptide-1, GLP-1) and glucose-dependent insulinotropic peptide (GIP). Both of these gut

    hormones constitute the class of molecules referred to as the incretins. Incretins are molecules

    associated with food intake-stimulation of insulin secretion from the pancreas.

    GLP-1 is derived from the product of the proglucagon gene (gene symbol = GCG). This

    gene encodes a preproprotein that is differentially cleaved dependent upon the tissue in which it

    is synthesized.

    Glucose-dependent insulinotropic polypeptide (GIP) is a key incretin hormone, released

    from intestine after a meal, producing a glucose-dependent insulin secretion. While, the GIP

    receptor (GIPR) is expressed on large neurons, and is synthetised in a subset of neurons in the


    It is also important to explain tthe role of aminoacids in the secretion of insulin. Specific

    amino acids may acutely and chronically regulate insulin secretion from pancreatic -cells in

    vivo and in vitro by altering the b-Cells membrane potencial. Mitochondrial metabolism is

    crucial for the coupling of glucose, alanine, glutamine and glutamate recognition with

    exocytosis of insulin granules.

    Mitochondria generate ATP (the main coupling messenger in insulin secretion) and

    other factors that serve as sensors for the control of the exocytotic process. The main factors thatmediate the key amplifying pathway over the Ca

    2+signal in nutrient-stimulated insulin secretion

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    are nucleotides (ATP, GTP, cAMP and NADPH), although metabolites have also been

    proposed, such as long-chain acyl-CoA derivatives and glutamate. In addition, after chronic

    exposure, specific amino acids may influence gene expression in the -cell, which have an

    impact on insulin secretion and cellular integrity. Therefore amino acids may play a direct or

    indirect (via generation of putative messengers of mitochondrial origin) role in insulin secretion.

    Arginine is the amino acid reputed to stimulate the highest production of both insulin

    and glucagon. L-arginine is a potent stimuli for insulin secretion from pancreatic b-cells.

    However, the precise molecular mechanisms of amino acid-induced insulin secretion have only

    partly understood.

    Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-

    secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets

    of Langerhans, has been shown to provide cholinergic input to the -cell in several species, but

    the role of autonomic innervation in human -cell function is at present unclear. It is known

    that the -cells of human islets provide paracrine cholinergic input to surrounding endocrine

    cells. Human -cells express the vesicular acetylcholine transporter and release acetylcholine

    when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by

    alpha cells in turn sensitizes the -cell response to increases in glucose concentration. It has

    beendemostrated by many scientists that in human islets acetylcholine is a paracrine signal that

    primes the -cell to respond optimally to subsequent increases in glucose concentration. [1]

    Acetylcholine not only increases the secretion of insuline by improving the -cells response, but

    also increases the granules movement. As show the pictueres below:

    Intracellular movement of secretory granules is a proximal stage in the insulin secretory

    cascade that ends in the release product from cells. The mechanisms underlying the control of

    this movement by acetylcholine has been investigated, thus trying to clarify how this NT acts.

    Acetylcholine activates intracellular movement of secretory granules as a result of

    muscarinic mobilization of intracellular Ca2 +.

    Acetylcholine has been reported to exert versatile effects on the secretory machinery of

    the pancreatic -cell. Hydrolysis of phosphatidylinositol 4,5,bisphosphate by activation of PLC

    via muscarinic activation positively controls insulin release by I P3-induced Ca2 +mobilization

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    Clockwise from left: the approach of vesicles to the membrane requires the formation of

    a Sec6Sec8 (exocyst) complex (DOCKING). Fusion of docked vesicles requires an elevation

    of Ca2+ concentration and a Ca2+ sensor such as synaptotagmin V/IX. Fusion is usually

    incomplete, releasing only a proportion of the vesicles soluble cargo and of vesicle membrane-

    associated SNARES such as synpatobrevin (brown) (84). Insulin is released as dimers with

    Zn2+ through a fusion pore likely to be less than ~4 nm across, whereas low molecular massspecies such as ATP (50) and lipid components of the vesicle membrane are likely to rapidly

    associate with plasma membrane lipids . Such "cavity recapture" events are terminated by the

    recruitment of dynamin-1 (RECAPTURE). Vesicles that have passed through a single round of

    exocytosis, but still retain adequate numbers of SNAREs, may then undergo further fusion

    events. Alternatively, vesicles may fuse with the endosomal network for destruction or may

    exchange membrane proteins and soluble cargo with maturing vesicles (EXCHANGE). The

    relative importance in the delivery to mature vesicles of membrane proteins (SNAREs, Sec6,

    phogrin, etc.) of de novo delivery at the TGN, and later delivery by the recycling mechanisms

    above, remains to be established.

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    1. Insulin signal transduction pathways.After insulin enters the bloodstream, it binds to an enzyme (Tyrosine Kinase) that acts as the

    insulin receptor (IR) and is located at the cell membrane.

    The insulin receptor (IR) is a tetrameric enzyme comprising two extracellular -subunits and

    two transmembrane -subunits. When insulin binds to the extracellular port of the IR the

    intrinsic tyrosine kinase activity of the -subunits of the IR is activated. This leads to an

    autotransphosphorylation of the IR where one -subunit phosphorylates the other on several

    tyrosine residues.

    The union of insulin to its receptor results in: decrease of the release and production ofglucose, and increase in the uptake of glucose. The uptake of glucose is caused by the

    translocation and exocytosis of GLUT4 storage vesicles and the activation of enzymes that

    activate the insulin transduction pathway and inhibit enzymes that slow/stop the insulin

    transduction pathway.

    The activity of the IR enzyme causes phosphorylation (activation) of two enzymes: Mitogen-

    activated Protein Kinase (MSP-Kinase) and Phosphatidylinositol-3-Kinase (PI-3K).

    The activation of MAP-Kinase leads to completion of mitogenic functions like cell growth

    and gene expression.

    The activation of PI-3K leads to the main insulin transduction pathway which intervenes in

    the completion of metabolic functions such as synthesis of lipids, proteins and glycogen. The

    PI-3K pathway separates the GLUT-4 vesicle (the main glucose transporter) from the glucose,

    and sends the vesicle to the plasma membrane while the glucose is sent to the mitochondria to

    produce ATP or is stored as glycogen. The steps of this pathway are:

    1. The insulin receptor substrate (IRS1/2) recruits to the plasma membrane the class Iphosphoinositide 3 kinase which is activated by this recruitment (PI3K is a

    heterodimeric protein composed of a p110 catalytic and a p85 regulatory subunit

    which binds to IRS adaptor proteins).

    2. Class I phosphoinositide 3 kinase catalyses the conversion of PIP2 to PIP3, so themain product of PI3K is the PI(3,4,5)P3.

    3. The increase of PIP3 induces the recruitment and co-localizationof protein kinase B(PKB or Akt) and PDK1. Once recruited to the plasma membrane PKB/Akt is

    activated by the action of PDK1 and TORC2. The PKB/Akt substrates that link insulin

    signaling events to GLUT4 translocation are: AS160, TBC1D1 and PIKfyve.

    3.1.AS160: contains two phosphotyrosine binding domains (PTB), a GAP domainselective for Rab small GTP-binding proteins and seven potential PKB/Akt

    phosphorylation sites. To allow the translocation of GLUT4, multiple Rab

    proteins must be activated. The GAP domain of AS160 suppress the activity

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    of these proteins therefore preventing GLUT4 translocation. The

    phosphorylation of AS160 is thought to render the GAP domain inactive and,

    as a consequence, stimulate GLUT4 translocation.

    3.2.TBC1D1: its very similar to AS160 but they differ, particularly, in theirphosphorylation. Expression of AS160 predominates in both adipose tissueand slow twitch muscle fibres, whereas TBC1D1 is more abundant in fast

    twitch muscle fibres. TBC1D1 also plays an important role in the stimulation

    of muscle glucose uptake in response to contraction and activators of AMP


    3.3.PIKfyve: this protein binds to PI3P and phosphorylates PI3P to yieldPI(3,5)P2. This process might link plasma-derived PIP3 signals to intracellular

    PI(3,5)P2 production, and thereby to the control of GLUT4 trafficking

    between endosomes and the TGN, from where GLUT4 may feed into the GSV

    pool. The protein PIKfyve is affected not only by insulin but also by

    hyperosmotic stress, being the response independent of PI3-kinase and


    The PI-3K pathway is also involved in the process of glycoysis as it controls the

    phosphorylation, and consequently activation, of PFK-2 which is a potent stimulator of PFK-1,

    the main enzyme-regulating glycolytic flux. To sum up, the activation of the PI-3K pathways

    increases the uptake of glucose and is controlled by different enzymes (i.e. GSK-3 and PKA,

    which constrict its activity, AKT and P70, which stimulate the pathway). Insulin will, therefore,

    increase the action of AKT and P70, and inhibit the activity of GSK-3 and PKA.

    Also important is how insulin controls the GLUT4 translocation to the plasma

    membrane because this vesicle is responsible for passive diffusion of glucose in the cell. There

    are two models of how this process takes place:

    Dynamic retention: the GLUT4 pool is in dynamic equilibrium between the cell surfaceand intracellular compartments. In this model insulin signalling increases the rate

    constants for docking and fusion of GLUT4 with the plasma membrane to increase the

    amount of glucose present at the plasma membrane. GLUT4 translocates to the plasma

    membrane via fusion with it, via a cycling pool or a combination of both routes.

    Static retention: the GLUT4 is actively retained within a storage pool that does notcommunicate with other intracellular compartments in the absence of insulin. Thismodel requires that insulin enters deep into the cell to induce the liberation of GLUT4

    from the pool(s) into the cycling system and from there towards the plasma membrane.

    Due to the position of the pool, GLUT4 must transverse the cytoplasm before arriving

    to the plasma membrane, but data suggests that microtubules dont play an important

    role in the movement of GLUT4 vesicles to the plasma membrane.

    Therefore insulin has two effects on GLUT4, it regulates the release of GLUT4 from

    intracellular storage pools and it increases the docking and fusion of GLUT4 vesicles with the

    plasma membrane reducing the mobility of GLUT4 vesicles in order to allow the fusion of those

    vesicles that have docked. Consequently insulin translocates GLUT4 from the intracellular

    stores to the plasma membrane where it increases glucose uptake by up to 2-fold over basal


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    The signaling pathway that regulates the process of docking and fusion appears to be PI3K

    dependent, as several studies suggest that the stimulation of PKB/Akt activity at the plasma

    membrane is the step in the insulin-sensitive signaling that ultimately leads to fusion. However,

    the movement of GLUT4 vesicles to the cell periphery is independent of PI3K signaling. The

    plasma membrane is an important site of insulin action because many studies suggest that

    insulins main regulation effect is not on GLUT4 vesicles but on the plasma membrane, butmore investigation remains to be done in this direction.

    The activation of the IR also causes the stimulation of a second signal transduction

    pathways that, although it plays a minor role in insulin-stimulated GLUT4 translocation, its

    involved in the regulation of important processes such as cortical actin rearrangements, PI(3)P

    formation at the plasma membrane, recruitment of the exocyst complex to the plasma membrane

    via Exo70 and the inactivation of Rab31. The steps of this pathway, known as Wortmannin-

    insensitive pathway, are:

    1. Recruitment and tyrosine phosphorylation of c-Cbl to the activated insulin receptor viatwo adaptor proteins (APS and CAP).

    2. c-Cbl recruits the adaptor protein CrKII and the guanine nucleotide exchange factorG3G to lipid rafts (where c-Cbl is attached) C3G specifically activates the small GTP

    binding protein TC10.

    In order to decrease the production of glucose in the cell, insulin indirectly activates PP-1. It

    is an enzyme that dephosphorylates and activates the glycogen synthase therefore increasing the

    synthesis of glycogen, and it also dephosphorylates and inactivates the glycogen phosphorylase

    decreasing, as a consequence, the rate of degradation of glycogen to glucose and the level of

    glucose in the cell.

    In conclusion, insulin acts on several intracellular signaling pathways that regulate

    metabolic activity, gene transcription and cell growth, being its main function the stimulation of

    glucose uptake by cells. To know about the components, function and steps of these pathways is

    an important advance in medicine because it will allow us to regulate them in order to treat

    many diseases related to insulin.

    2. The metabolic effects of insulin.The pancreas secretes insulin and equimolar amounts of peptide C. Between 10 to 15% of

    insulin detected by radioimmunoassay (RIA) corresponds to proinsulin.

    The insulin concentration determined by RIA in fasting is from 5 to 15 uU / ml and 30

    to 75 uU / ml in the postprandial period. C-peptide has a peripheral concentration 10 times


    C-peptide levels in fasting is 2 to 4 ng / mL and postprandial 4 to 6 ng / ml.

    Measurement of C-peptide concentrations in fasting or post glucagon stimulation is a good

    expression of the synthesis and secretion of insulin, which can be measured even in patients

    who receive insulin, since the latter has no cross-reaction with peptide C.

    The half-life of insulin is 4.8 and the proinsulin is 17.5 minutes.

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    The degradation of insulin was performed in liver and kidney, but preferably in the liver

    and proinsulin C-peptide and the kidney. Insulin in a high percentage is captured in its first pass

    through the liver, but not C-peptide

    The catabolism begins with the breaking of the disulfide bridges by the action of

    glutathione insulintransferasa, then start proteolysis, releasing inactive peptides.

    The biological activity of proinsulin is 10% of insulin and C-peptide is completely


    Insulin receptors:

    The biological action of insulin is mainly done through its interaction with specific

    receptors. Alpha units are recognized, responsible for recognition of the insulin molecule and

    beta units, location within the membrane, with the function of transmitting the message to the

    intracellular effectors. The receptors are degraded and resynthesized continuously now been

    identified the gene responsible for its synthesis.

    The number of counter regulatory receptors are negatively by the concentration of

    insulin and its affinity is reduced by the action of other hormones, among which

    catecholamines, glucagon, growth hormone, corticosteroids, estrogens, progesterone and

    placental lactogen.

    It has been established that the maximum insulin bioeffects can be maintained even with

    a concentration of 10% of recipients.

    Post-receptor effects of insulin:

    Although not known the exact the effects of the interaction between insulin receptor-

    transport systems and enzymes effector, is postulated as the most likely mechanism of action

    units autophosphorylation and activation of protein kinase beta, which would have the effect of

    second messenger.

    The second messengers, activate and inhibit gene transcription and action of enzymes

    involved in the metabolism of substrates, induce translocation of proteins, stimulate protein

    synthesis and transport of glucose, amino acids and ions.

    Thus, insulin activates glucose transport across the cell membrane of adipose tissue and

    muscle. We have identified a transporter located in the interior of the cell called Glut 4, the

    synthesis and translocation to the membrane is insulin-dependent. The transporter Glut 4 is also

    glucose-dependent, showing a negative counter-circulating glucose levels.

    Insulin increases hepatic action of stimulating glucokinase gene transcription of the

    enzyme and directly activates pyruvate dehydrogenase, acetyl CoA carboxylase and laglicgeno

    synthetase. On the other hand, directly inhibits the intracellular lipase and phosphorylases

    responsible for the mobilization of endogenous substrates.

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    Diabetes mellitus is a group of metabolic diseases in which a person has problems with sugar

    levels in blood because theres an insulin resistance in the tissues or the body is partially or

    completely unable to produce. According to the American Diabetes Association, the

    classification of diabetes includes four clinical classes: Type 1 Diabetes, Type 2 Diabetes, other

    specific types of diabetes due to other causes and Gestational Diabetes Mellitus.

    1. Type 1 Diabetes MellitusIt is a metabolic disease in which beta cells of Langerhans Islets in the pancreas are

    destroyed and the body losses the capacity to segregate insulin, so there is an insulin deficiency.

    This diabetes is also known as IDDM (insulin dependent diabetes mellitus) or juvenile diabetes,

    because it usually appears before the age of 25 years old.

    Causes and factors

    The main cause seems to be an autoimmune reaction. This process of self-destruction

    starts several years before clinical features of diabetes are diagnosed in a routine blood sugar

    test. At birth, the beta cell mass is normal and then the destruction starts to develop because of

    the autoimmune process , however the effects are not noticeable until the 80% of the beta cell

    are destroyed.

    This autoimmune process starts due to the pathological situation of insulitis - an

    inflammation of the islets of Langerhans of the pancreas- . Pancreatic -cells become infiltrated

    by mononuclear cells, leading to inflammation. This lymphocyte infiltration can result in

    destruction of the insulin - producing beta cells of the pancreas without the involvement of the

    glucagon-secreting cells (- cells of Islets of Langerhans) as a result of the attack of

    lymphocytes T together with macrophages. Also B lymphocytes are involved in this process by

    producing autoantibodies against -cells.

    However, as collateral effects, the destruction of beta cells can impair the activity of

    cells. In turn this results in an impairment in glucagon production. In fact, surveys involved in

    the effect of the destruction of cells into cells have demonstrated that the production of

    glucagon is affected by two main things: the destruction of the sympathetic nervous system dueto the autoimmune mechanism and directly by the loss of beta cells, because some substances

    that they produce, such as GABA, Zinc and even insulin it-self ,have inhibitory effects on

    glucagon production.

    Genetic factors: Most individuals with type I diabetes have the HLA DR3 and / or the

    HLA DR4 haplotype. Refinemens in genotyping of HLA loci have shown that the haplotypes

    DQA1*0301,DQB1*0302 and DQB1*0201 are associated with type I diabetes .On the other

    hand haplotypes DWA1*0102,D1B1*0602 provide protection against type I diabetes.

    Environmental factors: some viruses (such as coxsachie virus and rubella virus), bovine

    milk proteins and nitrosourea compounds could be involved in the pathogenesis of diabetes but

    this has not been proven yet.
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    Idiopathic diabetes type I

    Diabetes type I can be idiopathic, meaning, that there isnt a known cause. This

    idiopathic diabetes is mostly present in African or Asian people. This form of diabetes is

    strongly inherited and has permanent insulinopenia (insufficient production of insulin by the

    pancreas) and it is prone to ketoacidosis without antibodies against -cells.

    As we can see, ethnical factors are also involved in diabetes type I, which supports the

    theory of genetic factor involved in the disease: in Asian and African people the incidence of

    diabetes is the lowest one and the Caucasian are the most affected people by DMTI.

    Singns and symtomes : Before DMT-I is diagnosed the hyperglycemia situation presents

    the following main effects-: polyuria with glycosuria (frequent urination with the presence of

    sugar in the urine), polydipsia ( increased thirst), polyphagia (increased hunger), weight loss,

    nausea , vomiting ,abdominal pain and tiredness.

    Diagnosis: The diagnosis of DMTI in a person who presents these symptoms includes:

    Presence of immunological-involved molecules against beta cells: islet cells antibodies(ICAs), autoantibodies against insulin (IAAs), autoantibodies against glutamic acid

    decarboxylase (GAD65) and against tyrosine phosphatases (IA-2 and IA-2). These

    molecules are part of beta cells.

    Hyperglycemia and low blood insulin levels: the blood sugar under fasting condition ismore than 126 mg/dL and the aleatory blood sugar (that means, without fasting

    conditions) reaches more than 200 mg /dL.

    Glycated proteins and glycated haemoglobin: the excess of sugar causes the glycation ofdifferent proteins in the body. Glycated haemoglobin is the most easy way to check

    what level of hyperglycemia has suffered or suffers the patient.

    Most of the symptoms can lead to a diabetic ketoacidosis, which is a dangerous situation

    due to hyperglycemia that is characterized by an abundant present of ketone bodies in the urine

    and blood. This for instance, can cause abdominal pain and hyperventilation even leading to

    death if the treatment is not given in time.

    Treatment: Objectives in the treatment of any kind of diabetes are orientated to have a

    good management of the disease until arriving to the most permanent state of euglycemia

    (balanced and no pathological blood sugar levels ), to avoiding future complications (specially

    micro and macrovascular complications) and to live a life as normal as possible. The treatment

    of any diabetes have three points in common: diet, exercise and self-controlling of blood sugar

    levels. When it comes to diet and self-monitoring of blood sugar levels, treatments are different

    between DMTI and DMTII.

    Diet: The diabetic type I diet consists on a balanced diet, with the correct caloric support toensure a correct growth and development, without any lack of nutrients. The most

    important issue here will be the distribution of carbohydrates during the day. This

    distribution has to be personalized and have to take into account the number and type of

    insulin injections during the day. The nutritional indications in diabetes type II will be

    discussed later.

    Exercise: It helps in the maintenance of correct blood sugar levels ( controlling thuseuglycemia) and also preventing overweight.

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    Self-monitoring of blood sugar levels: In the case of DMTI this is the most importantmeasure because ,if there is something that really characterize diabetes type I, is the insulin

    dependence . Controls of blood sugar levels will be done with a blood-glucose metters,

    that offers an easy and fast way to have an idea of blood sugar, with a very low margin of

    error in calculation. From 3 to 6 times per day, the diabetic patient will need to check his

    blood sugar in order to adjust insulin doses.

    Hypoglycemia situation

    On the other side, a diabetic patient can present a hypoglycemia situation, showing a so

    low blood sugar levels (under 50 mg/dL) that can cause the diabetic comma , leading even to

    death. This hypoglycemia condition can be achieve by injecting an extra doses of insulin, a hard

    or prolonged exercise or under fasting conditions, also an inadecuate balance between meals and

    insulin injected can provoke hypoglycemia.

    Hypoglycemia will be treated depending on its gravity: once the first symtomes appear

    (sweating, sickness, hunger or nausea) the patient must take some high-sugar containing food,

    as juice, sugar drinks or plain sugar. In severe hypoglycemias, the patient can convulsionate, so

    he will need an injection of glucagon hormone (it has been discussed above the effect in

    glucagon secretion in diabetes type I) very quickly. So, glucagon is essential to revert extreme

    hypoglycemia situations.

    Long term consequences and effects

    The bad control of glucose levels in blood has several important effects, due mostly to

    the glycation of proteins what in turn leads to general problems such as: nerve damaging

    (neuropathy), mainly in peripheral nerves; macrovascular diseases ( the excess blood sugar canprovoke atherosclerosis, which can lead to a bad circulation, and cerebro-vascular problems,and

    also to heart arrest); microvascular diseases (the damage in capillaries and the increasement of

    density in blood sugar can cause escapes of sugars to the extracellular place, so black stains can

    appear in the skin; vision diseases (over all retinopaties), renal complication, feet ulceras,

    sexual complications.

    Future treatments and studies which are orientated to cure the disease

    Treatments are underway investigated following two different ways:

    Treatment of diabetes type I with stem cells: Nowadays, surveys with stem cells are beingdone. The transplant of stem cells of one patient to another one need an immunosupressor

    treatment, due to the rejection typical of an exogenous protein, so, this is a problem. But,

    most recently studies use the autotransplant that is ,stem cells from the patient- to try to

    cure diabetes and the results, so far, are being positive, but a lot of questions remain yet

    under debate.

    Islets Langerhans transplants: A promising alternative to insulin injections is cellulartransplantation. In this case, the treatment consists in harvesting beta cells from a cadaver

    and in introducing them into the blood stream of the patient. However, this also needs an

    immunosupressory treatment because of the rejection and to the attack of the immunitary

    system to the new beta cells, which , in the long-term, are destroyed again.

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    2. Type 2 Diabetes Mellitus2.1.Background.

    Diabetes Mellitus: Diabetes Mellitus (DM) represents a heterogeneous collection of

    metabolic diseases of very different etiopathogenesis, characterized by the common featureof chronic hyperglycemia, there being two basic physio-pathological disorders underlying

    any etiologic type of DM: 1- a defect in insulin secretion, 2- a defective insulin action or

    both, either one predominating at a given time.

    Endoplasmic Reticulum (ER) Stress:Many disturbances of the cell homeostasis cause

    accumulation of unfolded-misfolded proteins in the ER, triggering an evolutionarily

    conserved response, termed the unfolded protein response (UPR). The initial intent of the

    UPR is to adapt to the changing environment, and reestablish normal ER function. These

    adaptive mechanisms enhance the protein folding capacity of the ER, and promote ER-

    associated protein degradation to remove misfolded proteins. Excessive and prolonged ER

    stress triggers apoptosis.

    Insulin resistance (IR) is defined as the decreased tissue response to insulin-mediated

    cellular actions and is the inverse of insulin sensitivity. The term insulin resistance, as

    generally applied, refers to whole-body reduced glucose uptake in response to physiological

    insulin levels and its consequent effects on glucose and insulin metabolism.

    The most accepted hypothesis for insulin resistance is that it develops as a consequence

    of the effects of inflammatory and hormonal factors, ER stress, and the accumulation of by-

    products of nutritional overload on insuling-sensing tissues, such as adipocytes, hepatocytes,

    skeletal muscle and pancreatic -cells.

    Deposition of amyloid is the single most typical islet alteration in type 2 diabetes, where

    almost all of the islets are converted to amyloid. The amyloid formed may cause -cell

    apoptosis and dysfunction of remaining cells.

    2.2. What is type II diabetes mellitus (dmII)?:Type II diabetes mellitus is classified by the WHO under Endocrine, nutritional and

    metabolic diseases category. Its code is ICD-10 E11. According to the American Diabetes

    Association, Type II Diabetes Mellitus (DMII) is one the 4 clinical presentations of diabetes.

    Formerly called adult-onset or non-insulin-dependent diabetes, is the most commonform of diabetes. It is a complex, polygenic disease characterized by both hyperinsulinemia

    and hyperglycemia, at least early in its pathogenesis. Therefore, the term type II diabetes

    Mellitus designates not a single disease, but a heterogeneous collection of hyperglycemic

    syndromes phenotipically and genotypically different.

    It is thought to be the result of an imbalance between insulin sensitivity and secretion,

    with failure of pancreatic -cells to compensate for the increased insulin requirement created

    by IR leading to hyperglycemia.

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    2.3. SymptomsOften, people with DMII have no symptoms at firsteven for years. The early

    symptoms of diabetes may include: bladder, kidney, skin, or other infections that are more

    frequent or heal slowly; fatigue; hunger; increased thirst; increased urination. The first

    symptom may also be: blurred vision; erectile dysfunction; pain or numbness in the feet orhands.

    2.4. PrevalenceDMII has already reached epidemic proportions, affecting over 200 million people

    worldwide, being its prevalence of 6% in most developed countries.

    2.5.Risk factors: gene-enviroment interactions predisposing to dmIIDMII is strongly influenced by inheritance. Unlike type I diabetes, where a large

    component of heritability is found within a single locus (the HLA region), the heritability of

    DMII appears to be composed of many variants, each of modest impact on disease.

    It appears that IR and secretory deficiencies are necessary, but not sufficient to reach the

    levels of hyperglycemia that yield a clinical diagnosis. On the other hand, there is

    overwhelming evidence for the influence of genetic factors combined with a permissive

    environmental setting that determines risk of development of DMII.

    Clearly, epigenetics play a role in insulin resistance and in turn in DMII development as

    can be appreciated, for instance, by the fact that 80% of those with DMII reach a Body Mass

    Index (BMI)>30 Kg/m2. Moreover, people with BMI>35 Kg/m

    2(morbid obesity) have 100-

    fold greater risk for developing DMII than those with BMI

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    IR and PD usually have no symptoms. People with a severe form of IR may show

    acanthosis nigricans (Dark patches of skin, usually on the back of the neck, but sometimes

    on other possible sites). Other typical signs of IR syndromes include hyperinsulemia or

    polycystic ovary syndrome.

    Two physiopathological defects underlie DMII: defective -cell function and insulinresistance. Therefore, pathophysiologically DMII may range from predominantly IR with

    relative insulin deficiency, to predominantly an insulin secretory defect with IR.

    In DMII, islets of Langerhans dont show autoimmune destructive changes as in type I

    Diabetes (DMI), although amyloid deposits are frequently found in the islets.

    Natural history of DMII is variable resulting from long duration of silent

    hyperglycemia, often clinically reveled by symptoms and signs of specific macrovascular

    (e.g .stroke) and/or microvascular complications such as renal disease, retinopathy or

    obesity, among others.

    Ketoacidosis of acute onset as a complication of trauma, surgery or infection is rare

    compared to its frequent incidence in DMI, being more frequent in particular clinical

    phenotypes of keto-prone DMIIparticulary in Afro-Americans.

    The cornerstone of DM diagnosis is the unequivocal demonstration of plasma glucose levels

    higher than those accepted as normal. In the absence of unequivocal hyperglycemia

    diagnosis must be confirmed on a later date by any of the following three methods:

    1. Fasting Plasma Glucose (FPG)>=126 mg/dl Fasting defined as no caloric intakefor at least 8 hours.

    2. Symptoms of hyperglycemia and a casual plasma glucose>=200 mg/dlCasual isdefined as any time of day without regard to time since last meal. The classic

    symptoms of hyperglycemia include polyria, polydipsia and weight loss.

    3. 2-h plasma glucose >= 200 mg/dl during an Oral Glucose Tolerance Test (OGTT):This test measures blood glucose after people fast for at least 8 hours and 2 hours

    after they drink a sweet liquid provided by a doctor or laboratory.

    A blood glucose level between 140 and 199 mg/dL means glucose tolerance is notnormal but is not high enough for a diagnosis of diabetes.

    Hemoglobin A1C measurement (A1C) or glycated hemoglobin is a form of hemoglobin

    that is measured primarily to identify the average plasma glucose concentration over

    prolonged periods of time. It is routinely used as a tool for a metabolic control of DM,

    although its utility in the diagnosis has been a matter of debate for a long time. The cut-off

    point accepted for the diagnosis of DM is set at an A1C value of 6.5%.

    2.7. TreatmentThe primary goals of hyperglycemic management should be different depending on

    whether the patient has primarily -cell deficiency (AKA insulin-sensitive DMII), or

    whether the patient has IR leading to -cell insufficiency (AKA insulin-resistant variant of


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    In the case of insulin-sensitive DMII, the treatment of choice is drugs which stimulate

    insulin secretion. If these patients have severe -cell deficiency, they may need insulin

    replacement therapy.

    The insulin-resistant variant of DMII is the most common type of DMII in Western

    societiesdirectly related with the increase in ovesity. The initial approach to regulating theplasma glucose is to implement life-style changes with dietary intervention and increased

    physical activity. The objective is to maintain A1C

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