Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler INSULIN SIGNALING Related Reading Chapter 14 391395 in Stryer 6m edition OBJECTIVES 1 Compare the structures of proinsulin and insulin 2 Describe the mechanism for stimulation of insulin secretion and synthesis by glucose by increasing intracellular calcium 3 Delineate the steps leading to activation of the tyrosine kinase activity of the insulin receptor including the role ofinterchain autophosphorylation oftyrosine residues 4 Describe the mechanism by which insulin elicits mobilization of GLUT4 glucose transporter protein to the plasma membrane in muscle and adipose tissue via IRS p85 and PI3K 5 Compare the causes of type 1 diabetes with type 2 diabetes and eXplain what is meant by insulin resistance in the type 2 form PHYSIOLOGICAL PREMISE Insulin receptor defects are rare autosomal recessive disorders characterized by intrauterine and postnatal growth restriction hyperinsulinemia elevated blood insulin abnormal glucose homeostasis and short life eXpectancy Most cases have shown affected patients to be either homozygotes genes on both chromosomes with the same defect or compound heterozygotes defects in two or more locations of the same gene on a single chromosome on mutations in the insulin receptor IR xsubunit Such mutations impair insulin signaling by decreasing IR eXpression on the cell surface andor IR affinity for insulin strength of insulin attachment One patient was identi ed with two mutant alleles of the IR gene allele any of the alternative forms of a gene that may occur at a given locus The maternal and paternal alleles each contained a point mutation results from a change in a single base pair in the DNA molecule caused by the substitution of one nucleotide for another causing structural alterations in the tyrosine kinase domain of the IR Bsubunit resulting in severe impairment of insulin action INSULIN OVERVIEW General Information The physiology and biochemistry of insulin are quite signi cant because insulin plays an important role in fuel metabolism by promoting anabolic processes and inhibiting catabolic ones predominantly in muscle fat and liver tissue Insulin promotes storage of fuels as glycogen from glucose triglycerides from fatty acids and proteins from amino acids An acute action of insulin is the stimulation of glucose uptake into muscle and fat cells hence blood glucose is lowered by insulin Insulin Processing Insulin is synthesized and released by the Bcells of the pancreas Glucose is the primary stimulant to secretion The insulin molecule includes two separate chains held together by disul de bonds These chains initially are contained within a single polypeptide chain preproinsulin that is processed to proinsulin Fig l and eventually to insulin The two chains are connected by a portion known as the Cpeptide that is removed before insulin is secreted but is released together with the insulin Fig 1 After secretion the Cpeptide is slowly cleared by the Insulin Signaling l kidney In type I diabetics Whose Bcells have been destroyed by an autoimmune response there is no Cpeptide in the urine The lack of insulin to store glucose causes the blood levels to become elevated This excess glucose reacts with hemoglobin to form glycosylated hemoglobin and re ect the longterm elevated blood glucose Eventually these can crosslink seriously impairing the oxygen carrying ability of the red blood cells N i S 2 518 PROINSULIIE l E COO INSULIN Figure 1 The structural features of proinsulin and insulin Insulin secretion Secretei lilililililillll Protein kinase C DAG IMMEDIATE SECRETION OF INSULIN f 5 INSULIN 6 Ca2 BIOSYNTHESIS 2 amp PROCESSING Glucose gt Calmodulin E 032 CaM kinase 4 Figure 2 Control of insulin synthesis and secretion by glucose CaM kinase is calmodulindependent protein kinase DAG is diacylglycerol Insulin Signaling 2 Insulin release is mainly initiated by glucose as a signal of high carbohydrate intake An increase in the plasma concentration of glucose is the most important physiologic regulator of insulin secretion Insulin secretion in response to glucose is biphasic This response is characterized by an immediate or rstphase that begins within 1 minute and lasts for 510 minutes followed by a more gradual prolonged second phase that terminates soon after the glucose stimulus is removed These two phases likely re ect the different intracellular pools of insulin one which is at the plasma membrane surface and the other that is distant from the membrane The model in Figure 2 shows the most common biochemical signal transduction pathway in the control of insulin secretion Glucose acts by entering the Bcell via the GLUT Z Fig 2 1 transporter protein As a consequence of glucose metabolism 2 two signals are generated ATP and diacylglycerol DAG The increase in the concentration of ATP in the cell causes the membrane to close potassium channels that normally facilitate the eXit of K This event activates special voltagesensitive channels that allow Ca2 to enter the Bcell 3 Calcium triggers the exocytosis of secretory granules that contain insulin and Cpeptide Calcium also binds to calmodulin that activates calmodulindependent CaM protein kinase 4 to promote insulin biosynthesis 5 As discussed in an earlier lecture DAG together with calcium activates protein kinase C 6 which plays a role in promoting insulin synthesis and processing as well In this way the signal from high glucose not only causes the secretion of insulin but also initiates the events needed to replace insulin that has been secreted from the cell INSULIN RECEPTOR Structural features ocsubunits Figure 3 The insulin receptor Insulin binding to the occhains transmits a signal through the transmembrane domain of the Bchains to activate the tyrosine kinase activity membrane E f Transmembrane Tyrosine 2 domain kinase E CYTOPLASM 39 I domain 0 O C C O O Bsubunits Insulin Signaling 3 The insulin receptor IR is an integral membrane protein that eXists as a dimer Each monomer contains an on and a Bchain subunit Fig 3 The ocsubunits link to each other and to the Bsubunits by disul de bonds and lie entirely on the eXtracellular side of the plasma membrane Although each ocsubunit contains a binding site for insulin binding of one insulin molecule decreases af nity for binding of a second molecule This reduced binding for the second insulin molecule is termed negative cooperativity and is opposite to what is seen for oxygen binding to hemoglobin see lectures for chapter 10 A cysteinerich domain likely is involved in the binding event The Bsubunits traverse the membrane having tyrosine kinase enzyme domains on the cytoplasmic side Interchain Autophosphorylation Insulin binding ultimately switches on the catalytic activity of the tyrosine kinase domain on each Bsubunit of the receptor Fig 4 The activated insulin receptor tyrosine kinase IRTK then propagates the insulin signal by catalyzing the phosphorylation of proteins in the cytoplasm After binding insulin the receptor undergoes a conformational change that causes the IRTK on the same half of the receptor to which insulin binds Fig 4 3 subunit L to become active Fig 4 steps 1 9 2 Once this IRTK is activated it phosphorylates via interchain autophosphorylation the tyrosine kinase domain on the opposing Bsubunit R steps 2 9 3 Phosphorylation of tyrosine residues on the latter Bsubunit R activates that tyrosine kinase domain which in turn phosphorylates the initially activated Bsubunit L steps 3 9 4 Thus phosphorylation of the tyrosine kinase domains leads to enhanced catalytic activity ofthe tyrosine kinase independent ofinsulin binding Extracellular 1 2 3 Insulin IRTK L IRTK R IRTK L binds activated phosphorylated phosphorylated 39 d i O 39 v Cytoplasm H insulin 39 ADPS Phos ho lation O 7 IRTK 1nact1ve ATPS ADPS Gambling IRTK R IRTK active Phosphorylatlon catalyzed by IRTK L Figure 4 Activation of the tyrosine kinase domains of the insulin receptor by insulin binding followed by interchain autophosphorylation Insulin Signaling 4 Inactivation of the insulin receptor may occur by phosphorylation of the receptor on serine residues by an as yet unidenti ed serine kinase Therefore control of the insulin receptor comes about by l insulin binding and subsequent dissociation 2 autophosphorylation of tyrosine residues activation and 3 phosphorylation of serine residues inactivation Cessation of these events also occurs with destruction of the insulin Insulin has a plasma halflife of 3 to 5 minutes and the liver and kidneys primarily degrade it INSULIN SIGNAL TRAN SDUCTION Actions of insulin Understanding how the insulin signal is transduced is a fundamental problem in medicine It is crucial to understand this cascade in order to diagnose and treat patients with noninsulindependent type 2 diabetes mellitus NIDDM In the absence of insulin tyrosine residues adjacent to the tyrosine kinase domains remain dephosphorylated The insulinreceptor compleX undergoes an activation sequence involving conformational changes and autophosphorylations on the Bsubunits of the receptor as described above This triggering of tyrosine kinase activity leads to phosphorylation of several cytoplasmic proteins that can carry the insulin signal into the interior of the cell Besides phosphorylating the Bsubunits of the receptor activated insulin receptor tyrosine kinase phosphorylates a variety of proteins including insulin receptor substrate IRS phospholipase C and SHC protein The responses can be divided into metabolic and growth mitogenic The principal metabolic responses include activation of the insulinsensitive glucose transport system in adipose and muscle cells and of protein phosphatase in most tissues Protein phosphatase is responsible for dephosphorylating a variety of regulated enzymes that have been phosphorylated in response to hormone signals from glucagon or epinephrine as will be discussed in later lectures Recall that glucagon or epinephrine activates adenylyl cyclase to produce cyclic AMP that activates protein kinase A Protein kinase A in turn phosphorylates various enzymes associated with pathways of carbohydrate or fat metabolism Growth responses are mediated following phosphorylation of SHC through nuclear processes that include increases in synthesis of DNA and mRNA culminating in augmentation of cell growth and replication and protein synthesis respectively Mechanism of insulin action via IRS The several mechanisms by which insulin signals are transduced remain incomplete The student should note that many details remain hypothetical so that the events may differ when reviewing different teth Con icting ideas on these mechanisms may re ect the fact that studies have been conducted in different systems so that the nal mechanisms of insulin action will depend on the tissue of interest Until such time that the picture is complete it is best to refer to all of these as hypotheses The sheer compleXity of insulin signaling is the main reason why it remains so difficult for researchers and clinicians to identify appropriate treatments of NIDDM IRS insulin receptor substrate is a principal target of the phosphorylated active IRTK insulin receptor tyrosine kinase Several IRS proteins have been identi ed that have different functions Phosphorylation of tyrosine residues on IRS by IRTK activates it Activated IRS functions in this pathway as a docking protein The active IRS can dockwith a variety of other proteins to trigger several responses For instance IRS2 probably has been linked to regulation of the insulinsensitive glucose transport system in adipose and muscle cells Fig 5 In this mechanism IRS after phosphorylation by IRTK Fig 5 1 docks with p85 to facilitate the interaction of p85 with phosphatidylinositol kinase PI3K Fig 5 2 PI3K then phosphorylates phosphatidylinositol to phosphatidyl 4phosphate that in turn activates protein kinase B Protein kinase B through an unknown mechanism signals the Golgi to mobilize GLUT4 protein that is stored in this organelle for trafficking to the plasma membrane Fig 5 3 Thus in the fed state via this mechanism insulin increases the capacity of muscle and adipose tissue to use blood glucose Insulin Signaling 5 Extracellular O GLUT 4 space active receptor PO Cytoplasm k ros1ne 1nase IRS ty PO OP tvrOH ATP 1 IRTK catalyzed ADP IRS 2 activated 7 by docking active IRS IRS 3336 tyrOP PIP tyrOP PIP3 2 3 activated PI3K catalyzes Q PIP3 formation that activates PDK PDK activates 4 PKB signals Golgi to traf c GLUT4 to GOLGI OCOOOO 1 O GLUT4 Figure 5 Hypothetical mechanism for insulin to mobilize GLUT4 glucose transporter protein to the plasma membrane in muscle and adipose tissue Abbreviations used IRS insulinreceptor substrate IRTK insulin receptor tyrosine kinase PI3K phosphatidylinositol kinase PKB protein kinase B Insulin Signaling 6 Further detail of the recruitment of GLUT4 transporters in muscle and adipose tissue cells is depicted in Figure 6 DGlucose enters these cells by carriermediatedfacilitated diffusion a process enhanced in adipose and muscle cells by insulin This involves a Vmax effect increased number of transporters rather than a Km effect increased af nity of binding This is accomplished after insulin binding Fig 6 step 1 by recruiting glucose transporters GLUT 4 protein from an inactive pool in the Golgi body These transport proteins are then moved step 2 to the plasma membrane They then bind and fuse with the membrane step 3 to become available for glucose transport step 4 Once the insulin receptor is inactivated step 5 the excess GLUT4 returns to the Golgi step 6 Step 5 receptor inactivation Q Step 6 Translocation Back to Golgi 4 Glucose Step transport l glucose Step 3 Binding transporter and fusion Step1 insulin binding and signal transduction Figure 6 Insulin stimulated glucose transport GLUT4 in adipose or muscle cells Glucose transporters are stored in Golgi and translocated to the plasma membrane in response to insulin binding IN SULIN DEPENDENT TYPE 1 AND INDEPENDENT TYPE 2 DIABETES In diabetic patients there is major problem processing fats and especially carbohydrates for storage Consequently these patients present with elevated blood glucose concentrations hyperglycemia and this excess sugar may spill into the urine glucosuria The glucose takes water with it so that diabetes is associated with frequent urination and the danger of dehydration Patients with type 1 diabetes are also susceptible to onset of ketoacidosis overproduction of ketone bodies to be discussed in lecture 31 if they do not take insulin injections regularly Despite similarities in symptoms there are important differences between type 1 diabetes and type 2 diabetes Type 1 generally appears in childhood though there are a signi cant number of cases where the disease appears between the ages of 20 and 30 In type I diabetes the Bcells of the pancreas are destroyed by an autoimmune selfdestruction response Consequently the individual loses the ability to produce insulin and must be maintained with insulin injections for the rest of their life Hence the disease is termed insulindependent and is also referred to as juvenileonset because most cases appear in childhood Patients who fail to inject their insulin as prescribed face severe longlasting side effects due to the elevated blood glucose that can even non enzymatically attach to hemoglobin interfering with its function Insulin Signaling 7 Type 2 diabetes is often referred to as adultonset though there has been an alarming increase in the appearance of this disease in teenagers The initiating event in type 2 diabetes is generally the failure of tissues such as muscle and adipose tissue to decrease their ability to respond to insulin despite normal or even elevated amounts in the blood This is termed insulin resistance because the cells are resisting the effects of insulin This disease is generally insulinindependent because the pancreas continues to produce insulin However as the disease progresses the Bcells of the pancreas may lose some function because of their constant overstimulation by elevated blood glucose Overstimulation generally results from the intake of dietary excesses of carbohydrates so that obesity often precedes the onset of type 2 diabetes The alarming increase of type 2 diabetes observed in teenagers relates to unhealthy life styles in which teenagers are overweight This problem may be a particular issue in certain ethnic groups because of a propensity to become overweight Because the pancreas still produces insulin this ability generally prevents the development of ketoacidosis that may be seen in type 1 diabetics Table 1 Comparison of Type 1 and Type 2 Diabetes Mellitus increase in on and 8 cells Characteristic Type 1 Type 2 childhood and young adulthood middle and old age though now AGE AT ONSET but in rare instances may be as old starting to appear in preteenage as late 40s patients all races high incidence in Native RACE predominantly Caucas1ans Americans Blacks FAMILY PHSTORY OF 95 0 DIABETES MELLITUS rare common KETOSIS PRONE yes no unless insulindependent OBESITY rare common 80 MECHANISM autoimmune destruction variable often unknown INSULIN SENSITIVITY sensitive resistant REQUIRES INSULIN yes about half of the patients FIRST FUNCTIONAL decrease in insulin secretion decrease in res onse to insulin ABNORMALITY 1 INITIAL PATHOLOGY Ez slilmns5 reducnon 1 B39ce little or none amyloid deposition brosis LATE PATHOLOGY absence OfB cells Bcell mass normal or moderate reduction Insulin Signaling 8
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