You are on page 1of 28

PSIO 303A Test 1 1.

. Describe the concept of the Metabolic Syndrome and its consequences in increasing the risk of cardiovascular disease. The condition in humans where there is a clustering of specific pathologies that represent an increased cardiovascular disease risk, i.e. increased probability of suffering a heart attack or stroke. This can be referred to as Syndrome X, Insulin Resistance Syndrome, and/or CardioMetabolic Syndrome. 2. Discuss the core components of the Metabolic Syndrome (insulin resistance, hyperinsulinemia, glucose intolerance, visceral obesity, dyslipidemia, and hypertension) and recognize the associated components that also negatively impact cardiovascular disease risk. A. Core Components of the Metabolic Syndrome Insulin resistance (in skeletal muscle, liver, adipose tissue) Hyperinsulinemia Glucose intolerance and type 2 diabetes (AKA hyperglycemia) Visceral obesity Dyslipidemia Essential hypertension B. Additional Components of the Metabolic Syndrome Adipocyte dysfunction: dysregulation of adipokine secretion Accelerated atherosclerosis: increased cardiovascular morbidity and mortality Endothelial dysfunction: dysregulation of microcirculatory responses Renal dysfunction: micro- or macroalbuminuria Fatty liver: hepatic steatosis Inflammation: increased C-reactive Protein (CRP) and other inflammatory markers Hypercoagulability: increased fibrinogen and plasminogen activator inhibitor-1 (PAI-1) 3. Describe the developmental history of the core components of the Metabolic Syndrome, and defend the concept that insulin resistance and the compensatory hyperinsulinemia are early defects that can lead to other pathologies that constitute this syndrome.

Insulin has a regulatory role in the transportation of cations across the cell membrane. Elevated circulating insulin levels, such as in the case of hyperinsulinemia, cause intracellular sodium concentrations to increase, and intracellular potassium concentrations to decrease. This alteration in cation concentrations both intracellularly and extracellularly contributes to hypertension. Since hypertension is related to all other conditions of metabolic syndrome it can then be extrapolated that cation transport is ubiquitously associated with obesity, dyslipidemia, and glucose intolerance as well. In type 2 diabetes, the cells of the body become resistant to the effects of insulin as the receptors which bind to the hormone become less sensitive to insulin concentrations resulting in hyperinsulinemia and disturbances in insulin release. With a

PSIO 303A Test 1 reduced response to insulin, the beta cells of the pancreas secrete increasing amounts of insulin in response to the continued high blood glucose levels resulting in hyperinsulinemia. In insulin resistant tissues, a threshold concentration of insulin is reached causing the cells to uptake glucose and therefore decreases blood glucose levels. In insulin resistance regarding dyslipidemia, the body has to deal with the burden of excessive fatty acids, which may build up, in the liver as TG (triglycerides). The liver has 3 options for handling excess TG: (a) store it (b) burn it through beta-oxidation in mitochondria and (c) export it by synthesizing very low-density lipoproteins (LDL- a particularly atherogenic lipoprotein promotes the formation of fatty plaques that can occlude the artery and impair blood flow). Chylomicrons, also abounded with TG, are formed in the intestine after eating HOWEVER these should clear within a few hours after eating and should not be found in a fasting blood sample. Excess fat in muscle, liver, and visceral adipose tissue and muscle indicates insulin resistance. This is why dyslipidemia is traditionally comprised of excess plasma TG and free fatty acids both in fasting state and after meal (PP = post-prandial, after a meal), decreases in plasma HDL- cholesterol and post-heparin lipolytic activity (PHLA, a decreased ability to catabolize lipids in response to the drug heparin, which would contribute to excess lipid levels in the blood), and the small, dense LDL. This dyslipidemia will contribute to both the exacerbation of insulin resistance and the increased risk of a cardiovascular event (heart attack or stroke). Hyperinsulinemia has also been shown to "play a role in obese hypertension by increasing renal sodium retention" (elevations in both systolic blood pressure (>135 mm Hg) and diastolic blood pressure (>90 mm hg). 4. Define what is meant by the term normal glucoregulation, and describe how dysfunctions in the regulation of whole-body glucose homeostasis can be evaluated. In an oral glucose tolerance test (OGTT), 75 g of glucose are ingested. Normal glucoregulation should show that, after a night of fasting, blood glucose levels should be 90 mg/dl (5mM), and after an OGTT, blood glucose levels should return to normal after about 2-3 hours. In impared glucose tolerance (IGT), blood glucose levels will show that 140 mg/dl > [IGT] > 200 mg/dl and type 2 diabetes displays a 2 hour blood glucose level above 200 mg/dl. 5. Distinguish the specific physiological contributions of the major organ systems involved in wholebody glucose homeostasis, including skeletal muscle, the liver, the pancreas, adipose tissue, and the hypothalamus.

Skeletal Muscle- (~40% of body mass in humans), it is major tissue responsible for peripheral

PSIO 303A Test 1 disposal of glucose in response to a glucose load during a bout of exercise. Glucose transport into the muscle cell is controlled by insulin and muscle contractions. Glucose transport is the ratelimiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. Glucose transporter proteins mediate the transport of glucose across the muscle cell plasma membrane; two isoforms are expressed in muscle (GLUT1 & GLUT 4). Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT 4 transporter (but not GLUT 1) from an intracellular pool to the plasma membrane. The transported glucose can be stored as glycogen or oxidized to produce ATP. Liver- The liver can take up glucose from the hepatic-portal circulation (blood from gastrointestinal tract and spleen to liver) as well as from the general venous circulation and is therefore a site of glucose disposal; a more critical aspect of the liver in whole-body glucoregulation is the capacity of this organ to secrete glucose, a process called hepatic glucose production (HGP). Glucose production by the liver is mediated by a) the breakdown of glycogen stores (glycogenolysis) and b) the synthesis of new glucose molecules (gluconeogenesis). This is regulated primarily by the actions of insulin and glucagon. This prevents hypoglycemia Pancreas- has both exocrine and endocrine components; the endocrine consists of two cell types that are critical in normal glucoregulation are the -cells and the -cells. The - cells can be stimulated to synthesize and secrete the peptide hormone insulin, which we have seen is a critical factor for the stimulation of glucose transport in skeletal muscle (and in fat cells as well). On the other hand, the -cells synthesize and secrete the peptide hormone glucagon, which has critical effects on the liver for the regulation of hepatic glucose production. Adipose tissue- this is the site of insulin-stimulated glucose transport with glucose carbons being stored as TG or glycogen. More importantly however, adipose tissue contains adipocytes that are a critical site of the synthesis and secretion of several key regulatory factors called adipokines (or adipocytokines). These are secreted into circulation and can be composed of both positive and negative effects: Some adipokines that elicit positive cardiovascular and metabolic effects such as adiponectin and leptin, while others elicit negative cardiovascular effects including TNF-alpha, resistin, interleukin (IL-6), plasminogen activator inhibor-1 (PAI-1), and angiotensinogen Hypothalamus- Specific neurons in the hypothalamus are sensitive to nutrients (such as glucose or FFAs) and hormones (such as insulin or certain adipokines)

6. Summarize the long-term physiological consequences of impairments of whole-body glucoregulation in the Metabolic Syndrome (i.e., diabetic complications), and how oxidative stress can contribute to their development. Long-term hyperglycemia will have deleterious effects on numerous organ systems and increase risk of CV disease and other complications including dysfunctions in the eyes (retinopathy), the kidneys (nephropathy), peripheral neurons (neuropathy), and peripheral blood vessels (vascular dysfunction). One mediator of these effects is oxidative stress: excess formation of reactive oxygen species (ROS) and the formation of advanced glycation end-products (AGE). 7. Understand the general concept of signal transduction as a fundamental mechanism for cell-cell communication and for the modulation of the functionality of the target cell. External signals -> cell surface receptors -> intracellular second messenger systems -> cellular responses; external signals include odorants, metabolites, ions, hormones, growth factors, and neurotransmitters; Mentioned in class: also direct cell-to-cell transfer via pores called connexins

PSIO 303A Test 1

8. Describe the general mechanisms by which plasma membrane receptors initiate a signaling event in a cell and how the functionality of intracellular proteins can be modified by a change in phosphorylation state. The phosphorylation on serine, tyrosine, or threonine residues on the cellular target is mediated by the activation of a specific kinase, while the dephosphorylation of this cellular target is mediated by the activation of a specific phosphatase. In some cases, the phosphorylation of the enzyme increases its catalytic activity, while in the case of other enzymes the phosphorylation process decreases that enzymes catalytic activity. 9. Appreciate the concept of the signal amplification: how a large cellular event can be induced in response to a small initial extracellular signal.

PSIO 303A Test 1 10. List the four different types of receptors that exist in cells, and describe the detailed mechanisms for the functions of the three categories of plasma membrane receptors covered in class (ligand-gated ion channels, G protein-coupled receptors, and catalytic receptors). Ligand-gated ion channels integral membrane proteins that have extracellular and intracellular side, interact with ion channels or receptor itself is ion channel- either way, it opens ion channels that fluxes concentration and the ion is then the secondary messenger. Signaling molecule binds to the ionotropic membrane receptor that controls its opening and closing of the ion channel o Example of this type of channel in endocrine pancreas beta cells with K+ and Ca2+ o The neurotransmitter acetylcholine (Ach) acts on post-synaptic cholinergic receptors linked with Na channels, critical for depolarization of skeletal muscle G protein-coupled receptors (GPCRs) This is a very large class of integral membrane proteins that, when bound by the ligand, interact with and activate an intermediary heterotrimeric (alpha, beta, and gamma) complex called a G-protein (GTP-binding protein), which subsequently modulates a physically separate enzyme or ion channel. The product of this acts as the initial second messenger o Example of this type of receptor are the beta-adrenergic receptors affected by Epinephrine and norepinephrine and the alpha-(sub2)-adrenergic rectors, and the glucagon receptor o http://www.youtube.com/watch?v=V_0EcUr_txk Catalytic receptor ligand binding either activates enzymatic activity of the receptor itself or initiates a series of events that, through protein-protein interactions, causes the activation of an enzymatic complex. The substrate of the activated enzyme is the second messenger. Nuclear receptors (ignore until 303B, just know it exists)

PSIO 303A Test 1 11. Distinguish the different effectors with which G proteins can couple and mediate different intracellular effects.

(See video link above) The final step in this sequence of events is the hydrolysis of GTP (stimulated by members of the RGS family; RGS = Regulators of G protein Signaling), which then allows reassembly of the heterotrimer. G proteins can couple to various effectors: There can be interactions with and activation of adenylate cyclase (AC) via an s subunit (also known as Gs) (Fig. 4.13A-left), leading to activation of protein kinase A (PKA) (Fig. 4.13B-right). If the G protein is i (Gi), this will lead to a decrease in the activity of adenylate cyclase (Fig. 4.13A-left), a reduction in cAMP levels, and lower PKA activity. So Gs will activate AC which means MORE cAMP production means INCREASE in PKA. Gi activates AC, LOWERS cAMP production which means DECREASE PKA

There can be interactions with a phosphodiesterase (PDE) via t (Gt), which will break down cGMP and inactivates cGMP dependent channels (Fig. 4.13C-left)- (see below). An example of this mentioned in class includes transducin in the retina; the light activates the subunit and will degrade cGMP to GMP, which is then broken to something the brain can detect.

PSIO 303A Test 1

There can be an interaction with phospholipase C (PLC) via q (Gq), ultimately leading to an increase in the activity of protein kinase C (PKC) and increased intracellular Ca2+ levels (Fig. 4.13D-right) and activation of Ca2+ regulated molecules, such as calmodulin. Basically activates PLC, cleaves PIP2 into DAG and IP3, then the DAG activates the Protein Kinase (PKC) and the IP3 stimulates CA2+ release There can also be interactions with and activation of ions channels via interaction with Gs (an example is 1-adrenergic receptors in myocardium and skeletal muscle, which are coupled with Ltype Ca2+ channels). Basically- 2nd messengers include IP3, PAG, and CA2+. 12. Describe the mechanism by which receptor tyrosine kinases are activated and modulate the functionality of cytosolic effectors.

PSIO 303A Test 1

Receptor guanylate cyclases: produce cGMP from GTP (Fig. 4.14A) Receptor serine/threonine kinases: (Fig. 4.14B) Receptor tyrosine kinases: (Fig. 4.14C) - INSULIN RECEPTOR Tyrosine kinase-associated receptors: interact with and activate cytosolic tyrosine kinases (Fig. 4.14D) Receptor tyrosine phosphatases: (Fig. 4.14E) In in general, a single exofacial subunit of the receptor binds the ligand and this leads to dimerization (IMPORTANT NOTE: in the case of the insulin receptor, this receptor already exists as a dimer, with the exofacial subunits covalently attached by disulfide bonds) The dimerization of the receptors induces autophosphorylation and activation of the receptor complex (i.e., increased tyrosine kinase activity). There is enhanced tyrosine phosphorylation of specific cytosolic proteins. This leads to activation of downstream effectors and ultimately modifies cell function The activation of the receptor will cease once 1) the ligand-receptor complex is internalized or 2) by the action of a phosphatase to dephosphorylate the receptor. 13. You should be comfortable with the concept that the disposal of systemic glucose by skeletal muscle involves both the vascular delivery of glucose to the skeletal muscle and the transmembrane transport of glucose into the myocyte, and know how insulin regulates both processes in highly specific ways. Vascular delivery of glucose: The first consideration is the control of the so-called nutritive blood flow to the skeletal muscle from the systemic circulation. This will involve (1) the output of blood from the heart (cardiac output), (2) the degree of vasodilation in the vascular bed supplying the skeletal muscle of interest

PSIO 303A Test 1 (vascular resistance), and (3) the number of capillary contacts to the skeletal muscle (capillary density). All of these can be regulated by endocrine factors and other adaptive responses to factors produced by the skeletal muscle. For example, insulin can act on the endothelial cells in arterioles feeding the capillary bed of the skeletal muscle tissue to activate nitric oxide synthase (eNOS), causing the production of nitric oxide (NO). NO diffuses to the smooth muscle cells and causes relaxation and vasodilation, thereby promoting the delivery of both glucose and the insulin itself to the capillaries and then to the muscle for use there (Fig. 6.2). An additional consideration here is the leakiness of the endothelium of the capillaries in contact with the muscle, i.e., how easily can glucose and insulin pass through the endothelium to the extracellular space of the skeletal muscle tissue?

Transmembrane delivery of glucose into the myocyte: Once glucose and insulin are delivered to the extracellular space of the skeletal muscle, the insulin can bind to insulin receptors that reside in the sarcolemmal membrane of the myocytes, initiating a series of events (referred to as the insulin signaling cascade) that will ultimately allow glucose to enter the cell and be metabolized. At the onset of this discussion of the transmembrane glucose transport process, we need to recognize that the majority of glucose transport into the myocyte under insulin-stimulated conditions is facilitated by the glucose transporter isoform GLUT-4. The insulin receptor is a heterotetramer, consisting of two extracellular subunits that bind the hormone and two transmembrane -subunits that possess tyrosine kinase domains on

PSIO 303A Test 1 the cytosolic side. The - and -subunits are joined by several sulfhydryl bonds to form the functional reeptor. Insulin binds to the cysteine-rich domains of the - subunits, thereby enhancing, via a specific conformational change, the tyrosine kinase activity of the - subunits. The insulin receptor tyrosine kinase can phosphorylate itself (autophosphorylation), resulting in increased tyrosine kinase activity, and can phosphorylate various intracellular substrates, thereby propagating the insulin signal.

An activated insulin receptor (IR) can associate with numerous cytosolic and membrane protein targets via tyrosine phosphorylation of targets (such as IRS) and via interactions between phosphorylated tyrosine of IR and SH-2-containing protein targets. The Interactions determine cellular response; these include alterations in glucose transport and glycogen turnover, protein turnover, lipid turnover, and gene transcription. These effects of activated insulin receptor and IRS include stimulation of glucose transport (see below) and enhanced glucose storage as glycogen, increased protein synthesis and decreased protein breakdown, upregulation of lipid synthesis, and alterations in the expression of several genes in the nucleus. We will focus our attention here on the role of IRS-1 in the activation of glucose transport (Fig. 6.4). IRS-1 is a docking protein (meaning it will physically interact with other molecules), and when phosphorylated on specific tyrosine residues (by the insulin receptor tyrosine kinase), IRS-1 will be able to physically interact with downstream effector proteins that possess Src homology 2 (SH2) domains. A critical protein that can dock with tyrosine- phosphorylated IRS-1 via its SH2 domain is phosphatidylinositol-3-kinase (PI3K). The activation of PI3K, and subsequent activation of several other downstream elements of this insulin signaling cascade (including the enzyme Akt, also known as protein kinase B, or PKB) ultimately produces signals that causes the translocation of intracellular vesicles containing GLUT-4 to move to and fuse into the sarcolemmal membrane.

PSIO 303A Test 1

14. Describe how insulin, acting via the insulin receptor and post-receptor insulin signaling, impacts the translocation of GLUT-4 glucose transporter molecules to the sarcolemmal membrane of the myocyte to allow an increase in glucose transport activity. As stated above, the activation of PI3K, and subsequent activation of several other downstream elements of this insulin signaling cascade (including the enzyme Akt, also known as protein kinase B, or PKB) ultimately produces signals that causes the translocation of intracellular vesicles containing GLUT-4 to move to and fuse into the sarcolemmal membrane. This translocation process increases the number of functional GLUT-4 molecules in the plasma membrane and enhances the capacity of the myocyte to facilitate the movement of glucose from the extracellular space into the cytosol by a facilitative diffusion. This specific insulin signaling pathway is referred to as the IRS-1/PI3K/Akt pathway, and has been the subject of hundreds of investigations in the past two decades. Once insulin disengages from the insulin receptor or the insulin-insulin receptor complex is internalized and degraded, specific tyrosine phosphatases remove the stimulatory phosphate groups from the tyrosine residues on IRS-1 and the signal transduction process reverses, allowing GLUT-4 to return to its intracellular sites and causing glucose transport to decrease back to the baseline rate. 15. Know the distinguishing kinetic features of the major members of the GLUT family of glucose transporter proteins (GLUT 1-4) and discuss the physiological significance for why certain GLUT isoforms are highly expressed in specific cells and tissues.

PSIO 303A Test 1

For the above chart, keep in mind the normal range is 5-10mM. So how does this kinetic profile fit with GLUT-4s physiological role? We have seen that GLUT-4 transporters are inserted into the plasma membrane when muscle cells are stimulated with insulin. When the GLUT-4 transporters enter the membrane and are exposed to blood glucose, they are effectively saturated and are, therefore, delivering their full capacity to transport glucose; there is no reserve activity. If a higher rate of glucose transport is required to deliver more substrate to these cells, more transporters must be translocated from inside the cell and be inserted into the membrane. Thus the physiological role of GLUT-4 is met though its high affinity for substrate, which delivers the full transport capacity of each transporter following its insertion into the membrane. On the other hand, GLUT-2 is a high capacity, low affinity transporter because of its high Km. Since it has such a high Km it is basically entrenched in the plasma membrane, whereas GLUT-4 needs to be trans-located in response to increased levels of glucose. GLUT-2 needs to be far from saturation because it needs to be able to respond to increased levels of blood glucose. So if the concentration of blood glucose doubles, then the ability to transport glucose into the cell via GLUT-2 also "nearly" doubles in response. This should make sense since GLUT-2 is found in pancreatic beta cells, which produce insulin. GLUT-2 can adapt to increased levels of glucose, which will in turn allow the insulin response to be proportional, in normophysiological conditions. 16. Understand the concept that insulin resistance of skeletal muscle glucose disposal can arise from specific defects in both the vascular delivery of glucose to the skeletal muscle tissue and in the transmembrane transport of glucose into the myocyte. Vasoconstriction impairs delivery of glucose and insulin to the skeletal muscle. Insulin resistance in the endothelial cells can cause less activation of eNOS and less synthesis of NO. Reduced NO delivery to vascular smooth muscle cells result in vasoconstriction. There is much more evidence in the literature supporting specific defects in the insulin signaling cascade in the myocytes, primarily at the key initial sites of regulation of his cascade, namely the insulin receptor and IRS-1. This is further explored below (#17). 17. Describe the reciprocal relationship between tyrosine phosphorylation and serine phosphorylation in determining the functionality of the insulin receptor and IRS protein.

PSIO 303A Test 1 The binding of insulin by the exofacial -subunits leads to an increase in tyrosine kinase activity of the subunits. This initially causes tyrosine phosphorylation of the - subunits themselves (autophosphorylation), leading to an even greater tyrosine kinase activity of the insulin receptor. Furthermore, the activated insulin receptor will increase the functionality of IRS-1 by phosphorylating IRS1 on tyrosine residues. Therefore, tyrosine phosphorylation of the insulin receptor and IRS-1 is a positive modulator of these signaling elements. On the other hand, there are several serine residues on both the insulin receptor and IRS-1 that can be phosphorylated by a variety of enzymes that function as serine kinases. In general, serine phosphorylation of the insulin receptor decreases the tyrosine kinase activity of this protein by altering its three-dimensional conformation. This will impair the functionality of all downstream elements of the insulin-signaling cascade. Likewise, serine phosphorylation of IRS-1 will alter the three- dimensional conformation of IRS-1, and impair its ability to act as a docking protein with other signaling elements, such as the regulatory subunit of PI3K. This will also contribute to defects in the functionality of downstream insulin signaling elements, ultimately leading to decreased translocation of GLUT-4 to the sarcolemmal membrane and reduced glucose transport activity 18. and several stress-activated kinases) in negatively regulating the functionality of the insulin receptor and IRS proteins, and discuss the downstream consequences of dysfunctions in these initial elements of the insulin signaling pathway.

Protein Kinase C (theta) isoform- Protein kinase C (PKC) is a serine kinase that is expressed as one of several isoforms. Classically, the activity of PKC is regulated allosterically by the lipid diacylglycerol (DAG) and the ion CA2+. However, there are novel isoforms (PKC) that can be positively regulated by long chain acyl-CoA molecules, otherwise known as free fatty acids (FFAs).When there is increased delivery of FFAs to the myocyte, these PKC isoforms can be activated and they, in turn, will act on the insulin receptor and IRS-1 to increase serine phosphorylation and decrease their functionality. Decreased IRS activation of PI3K will lead to lesser PIP3 which means lesser PDK, then AKT, then GSV (the GLUT4 sequestering vesicle), which means less glucose.

PSIO 303A Test 1 So basically: FFAs were elevated in normal rats by lipid infusion, leading to the activation of PKC, enhanced IRS- 1 ser phosphorylation, and reduced insulin-stimulated glucose transport activity. Selective inhibition of PKCcompletely abolished the IRS-1 ser phosphorylation and enhanced insulinstimulated glucose transport activity in muscle. These findings are actually very important in making the connection between dyslipidemia and insulin resistance. In dyslipidemic states, elevations in plasma FFAs (likely due to increased hydrolysis of triglycerides in fat cells and in skeletal muscle tissue) can have the effect of inducing overactivity of PKC isoforms in myocytes and causing or exacerbating insulin resistance through serine phosphorylation of the insulin receptor and IRS-1. Stress Activated Protein Kinase- Insulin-resistant states (such as the Metabolic Syndrome) are characterized by increased plasma levels of inflammatory factors, such as C-reactive protein (CRP) or interleukin-6 (IL-6). Moreover, the hyperglycemia and dyslipidemia of the Metabolic Syndrome can increase the cellular production of reactive oxygen species (ROS), including superoxide ion and hydrogen peroxides. All of these deleterious factors can activate several different serine kinases in the myocyte, including I kappa kinase- (IKK), members of the mitogen-activated protein kinase (MAP kinases) family c-Jun N-terminal kinase (JNK) and p38 MAPK, and a downstream insulin signaling factor known as glycogen synthase kinase-3 (GSK-3). These serine kinases, in turn, are all known to phosphorylate IRS1 (and in some cases the insulin receptor) on serine residues, thereby impairing the functionality of these insulin signaling factors (Fig. 7.5). recent research findings have demonstrated that selective intracellular inhibition of these various serine kinases with small molecule inhibitors is associated with enhanced insulin signaling at the insulin receptor and IRS-1 and with increased insulin stimulation of glucose transport activity. For example, work from our research group here at the University of Arizona (Henriksen, E. J., and M. K. Teachey. Short-term in vitro inhibition of glycogen synthase kinase-3 potentiates insulin signaling in skeletal muscle of Zucker Diabetic Fatty rats. This indicates that selective inhibition of GSK-3 (the predominant isoform in muscle) improves insulinstimulated glucose transport activity in insulin-resistant skeletal muscle from a genetically-obese rat model of the Metabolic Syndrome and type 2 diabetes (called the Zucker Diabetic Fatty rat). This action of the GSK3 inhibitor is associated with increased tyrosine phosphorylation of IRS-1 and decreased IRS1 serphosphorylation (Fig. 7.8), indicating that GSK-3 can phosphorylate IRS-1 on this serine residue and negatively impact the ability of the insulin receptor to activate IRS-1 by tyrosine phosphorylation. 19. Describe the major cell types expressed, the hormones secreted, and the important anatomical features of the endocrine pancreas that are critical for normal glucose sensing by this organ. Made up of islets of Langerhans (alpha) will produce glucagon. The beta will produce insulin (in the center of the islet). F Cell- secretes pancreatic polypeptide. Delta cells produce somatostatin. Blood flows through the center of the islet to the ending of the islet. Their position is important because the insulin could hit the alpha cells and influence glucagon; there is little retrograde movement that would allow the glucagon to hit the beta cells. (1) Humoral communication: there are factors that are delivered to the Islet cells either by the systemic circulation (the primary factor here is blood glucose) and by paracrine delivery of pancreatic hormones to other cells within the Islet itself (insulin will inhibit glucagon secretion, glucagon will enhance insulin secretion, and somatostatin will inhibit secretion of both insulin and glucagon). It should be emphasized that blood flow in the Islets of Langerhans is from the center region, which is rich in -cells producing insulin, outward past the -cells (and the -cells and F cells) to the periphery

PSIO 303A Test 1 of the Islet, effectively allowing insulin to act on -cells, but effectively preventing glucagon from acting on -cells in this paracrine fashion. (2) Direct cell-cell communication: gap junctions exist between the different cell types and can deliver hormones from one cell to another (producing inhibition or stimulation of the secretion the target cells product, as described above). (3) Neural communication: the Islet is innervated by both the sympathetic and parasympathetic divisions of the autonomic nervous system. One important aspect of this neural regulation of Islet cell secretory function is the role of the sympathetic nervous system: adrenergic stimulation of -cells via norepinephrine will enhance glucagon secretion and adrenergic stimulation of -cells will inhibit insulin secretion. A second important aspect of this neural regulation is the role of the parasympathetic nervous system: cholinergic stimulation via acetylcholine (Ach) of -cells will stimulate insulin secretion. Modulation of the sympathetic and parasympathetic neural inputs to the Islets will therefore alter secretion (and therefore action) of glucagon and insulin. In this case, binding of glucagon to the glucagon receptor (a GPCR that interacts with Gs) causes a stimulation of adenylate cyclase, increases cAMP production, activates PKA, and promotes insulin secretion. However, as mentioned above (Fig. 8.2), the paracrine delivery of glucagon to -cells (and its action as an insulin secretagogue) is limited by blood flow in the Islet of Langerhans being directed first to the -cells and then to the -cells.

PSIO 303A Test 1

20.

List the major steps of the mechanism for coupling glucose transport in a pancreatic -cell with the appropriate secretion of insulin from that cell. a. Glut-2 is ALWAYS in the plasma membrane (not like Glut-4 that undergoes translocation to skeletal muscle) b. Never any limitation to how much glucose can be transported from beta cell c. First metabolized by glycolytic pathway (critical step towards creating ATP, which will act on potassium channel and when ATP levels go up, this causes potassium channels to close (ligand gated ion channel) d. When potassium channel closes (normally moves down concentration gradient out of cell) positive charge will build up, will lead to depolarization of the membrane e. This will have effect on voltage gated ion channel, calcium. Whether it is open or closed depends on membrane potential. Calcium normally in cells is very low. When calcium channel is open the calcium can move down concentration gradient, elevated [ca2+] leads to exocytosis and release into blood insulin (calcium stimulated calcium release) this always causes movement of secretary granules to the membrane where the insulin can be released- the insulin that goes up in bloodstream will help regular glucose levels, which deposits glucose into skeletal muscle, and the whole process becomes baseline

PSIO 303A Test 1 21. Discuss the detailed mechanisms for coupling low or high extracellular glucose with the appropriate secretion of glucagon from a pancreatic -cell.

Glucagon is synthesized in the -cells of the endocrine pancreas first as a proglucagon molecule, and then as one of several proteolytic products, that can include glucagon-related polypeptides (GRPP) and a Cterminal fragment. It is noteworthy that other cell types, such neuroendocrine cells in the intestine called L cells, can produce alternative cleavage products from the proglucagon molecule, such as the glucagon-like peptide GLP- 1, which acts as an incretin (AKA- group of gastrointestinal hormones that cause an increase in the amount of insulin released from the beta cells of the islets of Langerhans after eating). Major regulators of glucagon secretion are glucose levels, certain amino acids, and sympathetic nerve activity to the -cells. These modulators of glucagon secretion are described below:

Neural Regulation of Glucagon secretion:

Amino acid regulation of glucagon secretion: It is known that certain amino acids (such as the nonessential amino acids) can act as glucagon secretagogues, stimulating the release of glucagon from the cells. However, investigations into this action of amino acids have demonstrated that the concentration of amino acids needed to stimulate glucagon secretion in the intact organism is likely unachievable under most physiological conditions, bringing the physiological relevance of this mechanism into question. Moreover, the molecular mechanism of the secretory action of these amino acids is not known.

PSIO 303A Test 1

22.

Describe how hormone secretion from -cells and -cells of the pancreas can also be modulated by specific types of neural input to these cells.

PSIO 303A Test 1 23. Describe the two phases for dysfunctions in the secretory capacity of pancreatic -cells in the Metabolic Syndrome, the first manifesting itself in a compensatory hyperinsulinemia and the second leading to a relative failure of the -cell. The initial phase in the development of the metabolic syndrome involves an increase in blood insulin levels over a period of time that is associated with the degree of insulin resistance measurable in these individuals. This is known as compensatory hyperinsulinemia, as the increased levels of insulin in the bloodstream at this stage can compensate for and overcome (at least partially) the decrease in the ability of insulin to regulate glucose levels in the body. While the insulin levels are still elevated relative to what they were before the insulin resistance developed during compensatory hyperinsulemia, they are not high enough to overcome this insulin resistance and this phase is therefore characterized by a relative -cell failure. This relative -cell failure is absolutely necessary for the development of hyperglycemia in both the fasting state and during a glucose challenge (both characteristics of type 2 diabetes). Insulin Excursions during meals, measuring plasma insulin:

The concept here is quite simple: if there is a greater glucose stimulus after a meal or during an OGTT, there will be a proportionately greater secretion of insulin in response to that stimulus [enhanced ATP production leading to more depolarization with K+ ion channels closing and influx of Ca2+, greater calcium means more exocytosis and greater secretion of insulin from beta cell]. In reality, at this stage, there is no - cell dysfunction per se. There is simply an upregulation of insulin secretion due to a greater glucose stimulus, in an attempt by the body to dispose of this glucose load in the face of skeletal muscle insulin resistance. 24. Discuss the cellular mechanisms underlying the compensatory hyperinsulinemia and the relative cell secretory dysfunction observed in the Metabolic Syndrome.

The insulin-resistant state in this initial phase of the development of the Metabolic Syndrome is not restricted to dysfunctional glucoregulation. Insulin is also a powerful inhibitor of the breakdown of triglycerides stored in fat cells (and in other tissues), a process known as lipolysis. If the inhibitory action of insulin on lipolysis is reduced, there will be accelerated production and release of the end- product of lipolysis, namely free fatty acids (FFA) (these are also known as non-esterified fatty acids, or NEFA). The initial relative elevation in plasma FFAs (a part of the dyslipidemia that characterizes the Metabolic

PSIO 303A Test 1 Syndrome) will have several effects on the body. We have already seen one deleterious effect of FFA in the lecture on insulin resistance of glucose transport in muscle- [i.e.: raises ROS which raises all serine kinases; involves protein kinase c theta (PKC-theta)] Of importance in the present discussion is the observation that the increased plasma FFAs will initially lead to an increase in insulin secretion from the cells, likely due to the enhanced mitochondrial oxidation of FFA in the mitochondria, leading to increased ATP levels, closing of K+ channels, membrane depolarization, opening of Ca2+ channels, and eventually enhanced insulin secretion. As we will see below, this stimulatory effect of elevated FFAs cannot be maintain over the long-term, and this point will mark the transition from the phase of compensatory hyperinsulinemia to the phase of relative - cell failure. [Oxidative Stress]: In the -cell, elevations in ROS are associated with mitochondrial dysfunction, which would decrease the ATP-producing capacity of the cell and lead to decreased secretion of stored insulin. There is also evidence that FFAs, in the presence of hyperglycemia and independent of their effects to produce oxidative stress, can inhibit the transcription of the insulin gene, leading to reduced cellular production of insulin (cell will have less to secrete). This down-regulation of insulin production can also be attributed to increased levels of inflammatory cytokines (especially adipokines such as TNF- and resistin) associated with visceral adiposity. Loss of -cells (apoptosis) can occur as well; it is a critical development that can contribute to the relative -cell dysfunction that characterizes the Metabolic Syndrome and type 2 diabetes. As with dysfunction of existing - cells, the apoptosis of -cells can be enhanced due to the oxidative stress associated with the long-term glucose intolerance (hyperglycemia following a glucose load) and elevated FFAs. Interestingly, the adipokine leptin, which has a critical function in feeding behavior and is elevated in conditions of obesity, has been causally linked with pro-inflammatory activity in the -cell, with increased -cell apoptosis, and with decreased -cell mass. While the process of apoptosis is not completely understood, it does involve the activation of intracellular enzymes called caspases, which are cysteine proteases critical to this process of programmed cell death.

25.

Defend whether or not a true dysfunction in the glucagon secretory mechanism in -cells of the

PSIO 303A Test 1 pancreas develops in the Metabolic Syndrome. There is no evidence in the literature describing a down-regulation of -cells in the progression of dysfunction of glucoregulation leading to the development of the Metabolic Syndrome. In fact, there is actually evidence that the post-prandial secretion of glucagon from the -cells is not suppressed in type-2 diabetes as it normally is in individuals with normal glucose tolerance. This exaggerated glucagon response will exacerbate the state of hyperglycemia (glucagon will release more glucose into the blood). There may also be an increase in SNS activity, increased NE delivered which, after enhanced Ca2+ released from ER, will cause glucagon secretion from ER. 26. Describe the basic mechanism whereby insulin stimulates glycogen synthesis and suppress glycogen breakdown and gluconeogenesis in the liver, and how insulin decreases hepatic glucose production. Overall insulin does three things: ENHANCES glycogenis (production of glycogen for storage of glucose), INHIBITS glycogen breakdown (glycogenolysis), and SURPRESSES gluconeogenesis. Enhancement of glycogen synthesis: Insulin causes rapid activation of IR and IRS. The IRS signals via two pathways:via PI3K and Akt to inhibit GSK-3 via MAPK and p90-S6 kinase to activate PP1G Both pathways activate GS and increase glycogen synthesis: this will inhibit HGP. Inhibition of glycogen breakdown: Insulin acts via IR/IRS/MAPK/p90-S6 kinase pathway to activate PP1. PP1 dephosphorylates and inhibits phosphorylase. This results in inhibition of glycogenolysis and reduced HGP. Suppression of gluconeogenesis: Insulin acts via inhibition of GSK-3 and activation of p38 MAP kinase pathways to suppress transcription of PEPCK and G6Pase (critical gluconeogenic enzymes). This lowers protein levels of PEPCK and G6Pase reduce gluconeogenesis and HGP. 27. Discuss the basic mechanism for the action of glucagon to stimulate glycogen breakdown and gluconeogenesis and suppress glycogen synthesis in the liver, and how glucagon enhances hepatic glucose production. Glucagon stimulates HGP by INHIBITING glycogenesis, ACTIVATING glycogenolysis, and ACTIVATING gluconeogenesis. The plasma membrane of the hepatocyte is rich in glucagon receptors. As was described in lecture 8, the glucagon receptor is a GPCR that interacts with Gs, causing stimulation of adenylate cyclase, increasing cAMP production, and activating PKA. The activation of PKA by glucagon will have two distinct effects, one rapid and the other longer-term, that will impact glucoregulation via the liver. Glucagon acts via the G(s)/adenylate cyclase/cAMP pathway to activate PKA. PKA phosphorylates and activates phosphorylase kinase, which then phosphorylates and activates phosphorylase. Enhanced glycogen breakdown increases HGP. In addition to enhancing glycogen breakdown, the activated catalytic sub- units of PKA will migrate to the nucleus and phosphorylate CREB at the CRE site. In concert with CBP, this will ultimately increase gene transcription and the protein expression of PEPCK and G6Pase. This is an important long-term mechanism of glucoregulation, as the contribution of increased glycogen breakdown to enhance HGP is only transient (as glycogen stores in the hepatocytes are finite and depleted fairly quickly). If the increased rate of HGP must be maintained for a period of one hour or more (such as

PSIO 303A Test 1 during prolonged aerobic exercise), this can only be achieved if the rate of gluconeogenesis is ramped up. 28. Defend the significance of the glucagon:insulin ratio as a key regulator of hepatic glucose production. The hormone insulin primarily directs the process of glycogen (the polymer form of glucose) synthesis, which is then stored in hepatocytes. It is important to keep in mind that under conditions where insulin secretion is stimulated (such as following a glucose load from a meal or during an OGTT), the paracrine action of increased insulin contacting -cells will help to decrease glucagon secretion. On the other hand, under conditions where insulin secretion is diminished (such as during a bout of exercise or with a -cell dysfunction), glucagon secretion can be enhanced due to the decreased inhibitory action of insulin and the increased sympathetic neural input to the -cells. The second role of the liver involves the ability of the hepatocytes to produce and export glucose into the blood stream, a process known as hepatic glucose production (HGP). As we will see below, there are two sources for HGP: via the rapid breakdown of stored glycogen (a process known as glycogenolysis) into glucose units and via the intracellular synthesis of new glucose molecules from precursors (a process known as gluconeogenesis). Remember, beta cells produce insulin and this can inhibit glucagon from the alpha cells; glucagon activates glycogenolysis and gluconeogenesis to increase HGP. This reciprocal relationship between the circulating levels and actions of insulin and glucagon will be critical for normal regulation of HGP under conditions when plasma glucose levels are far from the set-point. 29. Define the time course for excessive hepatic glucose production in the development of the Metabolic Syndrome. Gerald Reaven has provided important insight into the development of elevated HGP in the Metabolic Syndrome and type 2 diabetes. Using the euglycemic, hyperinsulinemic clamp in humans (this technique allows for the measurement of whole-body glucose disposal and can be used to assess HGP), Reaven provides evidence that HGP is not elevated above normal in individuals with the Metabolic Syndrome until these individuals are in the phase of relative -cell dysfunction (see right below), when insulin concentrations fall from their previously elevated levels in the compensatory hyperinsulinemia phase. According to Reaven, a critical metabolic alteration during this phase of relative -cell failure is an increase in the circulating level of FFAs, as the relative decrease in insulin would facilitate less inhibition of lipolysis (remember that insulin normally suppresses lipolysis, primarily in fat cells) The elevated FFAs could be taken up by the hepatocytes and used as an oxidative substrate, producing ATP, and mediating greater flux through the gluconeogenic pathway, which has several ATP-requiring steps. In other words, increased FFA availability due to a defect in insulins ability to suppress lipolysis would drive greater rates of gluconeogenesis in the liver and therefore increase HGP.

PSIO 303A Test 1 30. Discuss the contributions of defects in insulin secretion and action responsible for excessive hepatic glucose production in the Metabolic Syndrome. See above. Additionally: Activation of serine kinases goes up (including PKC isoforms, IKK, p38 MAPK, JNK, and GSK-3), raising Ser phosphorylation of IR and IRS, lowering Tyr phosphorylation of IR and IRS, there would be a lesser activation of PP1 (the enzyme that dephosphorylates and inactivates the enzyme phosphorylase, the rate-limiting step in this process), which lowers the suppression of glycogenolysis and gluconeogenesis, which then raises HGP. Also, the dysfunction of IRS-1 would also compromise the ability of insulin to suppress gluconeogenesis, as there would be diminished inhibition of the transcription of two critical enzymes involved in the gluconeogenic pathway, PEPCK and G6Pase. 31. Discuss the significance of the alterations in glucagon secretion and action that contribute to excessive hepatic glucose production in the Metabolic Syndrome. While there is no direct evidence that there are specific cellular dysfunctions in glucagon action in the enhancement of HGP, the following contributes to the increase in glucagon and HGP:

32. Defend whether the glucagon:insulin ratio is an accurate way of estimating increases in hepatic glucose production in the Metabolic Syndrome and in type 2 diabetes. In pathophysiological conditions (such as the Metabolic Syndrome and type 2 diabetes), the glucagon:insulin ratio would underestimate the increase in HPG, because it cannot take into account the insulin resistance at the cellular level in the liver. For example, there certainly could be a synergistic interaction between existing glucagon action and the defective insulin action, resulting in a markedly enhanced HGP. This again underscores the importance of the glucagon:insulin ratio when considering alterations in HGP in physiological (such as after a meal or during exercise) conditions. 33. Discuss the concept that central obesity is a key component of the Metabolic Syndrome, and that factors produced by fat depot (adipokines) are linked to the metabolic dysfunctions and increased cardiovascular risk factors displayed by individuals with this syndrome. Until about twenty years ago, adipose tissue was considered to have the primary function of acting as a storage site for lipid in the body, which could be mobilized and used as an energy source during times of caloric restriction. It was well known to health care professionals that an excess of adipose tissue, especially in the visceral region, increases the risk of a cardiovascular event: suffering a heart attack or a

PSIO 303A Test 1 stroke. While this remains a critical feature of fat cells in the body, we now know that adipose tissue can also act as an endocrine organ, secreting an array of chemical messengers, known as adipokines or adipocytokines, that can act on numerous tissues in the body in both beneficial and deleterious ways. An important concept, though, is that these factors derived from adipocytes can alter the functionality of target cells by signaling via plasma membrane receptors. In many cases, the degree of obesity correlates with the circulating level of various adipokines: this would be a positive correlation for adipokines that elicit negative cellular responses (such as TNF-, IL-6, PAI-1, and resistin) and would be an inverse relationship for some adipokines that produce beneficial cellular actions (such as adiponectin, but not leptin).

34. List the important regulatory mechanisms underlying the secretion, vascular transport, and cellular action of adiponectin, a beneficial adipokine. Adiponectin is transported in the plasma bound to a binding globulin (e.g. albumin) in order to enhance solubility and increase half-life. Transendothelial transport of adiponectin at target tissue (like skeletal muscle) is regulated. May be defective in insulin-resistant states.

There is cutting edge research that indicates that the transendothelial transport of adiponectin into the interstitial space of target tissues, such as skeletal muscle, may be impaired in conditions of insulin

PSIO 303A Test 1 resistance, indicating that this vascular defect in adiponectin transport may contribute to the development of the insulin-resistant state. There are 3 total receptors: AdipoR1, AdipoR2, and T-cadherin (which has no cytoplasmic region).

Other important cellular responses to this adiponectin/AdipR1/APPL signaling include increased fat oxidation, thereby decreasing the potential deleterious effects of FFAs on insulin sensitivity (see lecture 7) and enhanced nutritive blood flow to the skeletal muscle tissue due to increased production of the vasodilator nitric oxide (NO). 35. Describe the defects in adiponectin secretion and cellular action that are associated with the obesity- related insulin resistance of the Metabolic Syndrome. First, the synthesis and secretion of adiponectin from these central fat depots is decreased with increasing fat cell size (adipocyte hypertrophy underlies increased visceral adiposity). A role of increased FFAs in this down regulation of adiponectin synthesis and secretion has also been suggested. The decreased secretion of adiponectin will result in less circulating adiponectin available to act on target cells. Second, the possibility

PSIO 303A Test 1 exists that transendothelial transport of adiponectin from capillaries to the interstitium of target tissues may be compromised in conditions of enhanced central obesity. Finally, there is evidence that the expression of adiponectin receptors, including AdipR1 and AdipR2, is downregulated in obesity, possible due to the hyperinsulinemia that co-exists with obesity. This decrease in adiponectin receptors would diminish the ability of adiponectin to transduce signals that would otherwise enhance insulin sensitivity (this represents the so-called adiponectin resistance).

36. Describe in general terms the prevalence of hypertension in conditions of insulin resistance, including the Metabolic Syndrome. Increases in CO due to renal water retention or increases in the vasoconstriction of arterial vascular beds, thereby increasing TPR, could lead to a hypertensive state. definition of hypertension, with elevations of SBP above 135-140 mm Hg and DBP above 85-90 mm Hg being considered diagnostic thresholds. As shown in Fig. 2.4 revisited, the onset and worsening of hypertension in the Metabolic Syndrome occurs during the phase of compensatory hyperinsulinemia. A critical question would be: is there a mechanistic connection between hyperinsulinemia and hypertension? However, the most important findings were that the degree of SBP was positively correlated with

PSIO 303A Test 1

hyperinsulinemia and both SBP and DBP were inversely correlated with insulin sensitivity (i.e., the lower the insulin sensitivity (M-value), the worse the blood pressure readings). These data support (but do not prove) an interconnectedness between insulin resistance, hypeinsulinemia, and defects in blood pressure regulation.
37. Discuss the potential mechanistic contributions of hyperinsulinemia and sympathetic over activity to long-term adaptations in the kidney and the peripheral vasculature that can lead to a hypertensive state in the Metabolic Syndrome.

The increased SNS activity can, via an upregulation of output from adrenergic neurons to the kidney, cause increased renal Na and water retention, leading to greater blood volume, a greater CO, and therefore increased blood pressure. The sympathetic overactivity will also be associated with enhanced constriction of peripheral vascular beds, which increases TPR and arterial blood pressure. enal and vascular events independent of its effects mediated by sympathetic overactivity. The enhanced insulin can act at the kidney to upregulate Na and water retention, both via direct effects on the renal tubules and via an increased proliferation of the Na/H exchanger. This increased Na and water retention will increase blood pressure through an increase in blood volume. The chronic elevation in plasma insulin will have two additional effects. First, the hyperinsulinemia will induce a proliferation of vascular smooth muscle cells (VSMC) in the arterioles, producing a remodeling of that vascular bed. Second, the dyslipidemia (increased plasma levels of triglyceride and FFAs) associated with the insulin-resistant, hyerinsulinemic state will promote an

PSIO 303A Test 1 acceleration of the formation of atherosclerotic plaques in these blood vessels. This combination of a greater expression of VSMC and the development of atherosclerosis in these arterioles, over the long term (i.e., a period of years or decades), will result in blood vessels that are more rigid and have enhanced responses to vasoconstrictors (such as angiotensin II; see next section below), leading to an increase in TPR and MAP. 38. Defend the concept that sympathetic overactivity can lead to upregulation of the renin-angiotensin system and can contribute to hypertension in the Metabolic Syndrome. Neural activity arising from the SNS will have the effect of causing the release of an enzyme (renin) from specialized cells of the kidney called juxtaglomerular (JG) cells. Renin is a peptidase that cleaves a portion of the peptide sequence of a precursor molecule called angiotensinogen, producing an intermediate product called angiotensin I (ANG I). ANG I can then be acted upon by a different peptidase called angiotensin converting enzyme (ACE), producing a final, bioactive peptide called ANG II. ANG II can have two effects that impact blood pressure. The first effect of ANG II is on the adrenal cortex to produce the steroid hormone aldosterone, which acts on the kidney to promote the retention of Na and water, increasing CO. The second effect of ANG II is to induce vasoconstriction of arterioles (by binding ANG II receptors on the endothelium), increasing TPR, (which, again MAP=CO*TRP). In the Metabolic Syndrome, the sympathetic overactivity will induce an upregulation of renin secretion and ultimately of ANG II action, causing hypertension. Luckily, this type of hypertension can be corrected with inhibitors of the peptidase ACE (ACE inhibitors) or with drugs that directly antagonize the ability of ANG II to bid its receptor (ANG II receptor blockers, or ARBs). The newest class of anti-hypertensive drugs directly inhibits the enzymatic activity of renin, stopping this pathway at its initial step.

Remember that both actions of ANG II are associated with increased blood pressure- the increase in renal water absorption AND the increase in vasoconstriction.