Insulin signal transduction pathways | Endocrine physiology

Insulin signal transduction pathways | Endocrine physiology 

There are 2 fundamental signal transducing pathways by which insulin acts: 

1. Phosphotidylinositol-3 kinase or PI3-Kinase pathway 

2. Ras-MAP kinase pathway. 

Insulin acts by binding to its receptors which are present on the cell membrane of its target cell. Insulin receptors are enzyme linked receptors which have inherent tyrosine kinase enzyme activity. 

The insulin receptors are present as tetramers i.e they have 4 polypeptides or subunits which assemble to form the receptor. Two of these subunits are alpha subunits while the other two are beta subunits. Alpha subunits project outside the membrane i.e form the extracellular part of the receptor which has the binding site for insulin. The beta subunits traverse through the membrane  and protrude into the cytoplasm and has  intrinsic tyrosine kinase activity i  When insulin is not bound to the receptors, the tyrosine kinase is inactive. 

When the insulin binds to its receptors, the tyrosine kinase activity of beta subunits activates which then cross-phosphorylate tyrosine residues of each other. This receptor then binds to insulin receptor substrates.  Once that happens this insulin receptor substrate becomes a docking site for other kinases and adaptor proteins. 

 1. PI3-Kinase pathway: 

 Phosphotidylinositol-3 kinase  phosphorylates  phosphatidylinositol 4-5 biphosphate. For this, initially it binds to IRS. This brings it close to its substrate which is present on the membrane and also activates it by phosphorylation. Then, it phosphorylates, PIP2 forming phosphotidyinositol 3,4,5 triphosphate.  Now this triphosphate also acts as a docking site for 2 other kinases i.e  phosphoinositide dependent kinase 1 and Protein kinase B. Now  PDK-1 phosphorylates  protein kinase B or AKT thus activating it. 

It is this protein kinase B which now dissociates and moves into the cytoplasm which then causes insertion of GLUT 4 receptors on the membrane, activates glycogen synthase for conversion of glucose to glycogen, inactivates glycogen phosphorylase for preventing glycogenolysis and hence causes varied effects on metabolism. 

 2. RAS-MAPK pathway 

 Monomeric G-proteins (Ras) have the ability to bind GDP and GTP. In inactive state , they are bound to GDP while when active they bind to GTP just like our trimeric G proteins. For activating it,  insulin receptor substrate binds with another adaptor protein GRB2 which in turn binds and activates a Guanine exchange factor protein SOS  which replaces Ras GDP with GTP causing its activation. The activation of Ras inturn leads to activation of kinase cascade including Raf, MEK and then ultimately leading to the activation of MAPKinase.  After activation, MAP kinase translocates into the nucleus and phosphorylates many transcription factors that regulate expression of important cell-cycle and differentiation-specific proteins. So that’s how insulin has effects on cell proliferation, growth and differentiation. 

 In summary, insulin acts by 2 signal transduction pathways: PI3-Kinase pathway responsible for its metabolic actions and some survival actions also and Ras MAP kinase pathway responsible for its effects on cell proliferation, growth and differentiation.

Diuretics: Mechanism of action, uses | Renal Physiology | Pharmacology

Diuretics are substances which increase the flow rate of urine. Most of the classes of diuretics work by increasing both the solute as well as water loss.

 1. Carbonic anhydrase inhibitors: They inhibit the carbonic anhydrase enzyme present in epithelial cells lining the proximal convultae tubule. Thus, they decrease the reabsorptionof sodium as well as that of bicarbonate. Due to this, there is decrease in pH causing metabolic acidosis. They ae used mianly to block carbonic anhydrase enzyme in eyes especially in open angle glaucoma and in high altitude sickness where they prevent the development of metabolic alkalosis.

 2. Loop diuretics: Loop diuretics inhibit sodium potassium 2cl- transporter. This interefers with both the concentration as well as dilution of urine. They are very effective diuretics since they interfere with absorptiono f approximatelu 25% of sodium reabsorptionin nephron.

 3. Thiazide diuretics: These diuretics act on distal convulated tubule where they block sodium chloride symporter. They mainly interfere with dilutionof urine and not with concentrationo f urine. They are moderately effective diuretics which interfere with 5-10% of reabsorption of filtered sodium.

 4. Potassium sparing diuretics: They act on late distal tubule and collecting ducts. They act by inhibiting either epithelial sodium channels or mineralocorticoid receptors. They are not very effective diuretics as such but when combined with other diuretics, they help in preventing the development of hypokalmeic alkalosis.

 5. Osmotic diuretics. Osmotic diuretics interfere mainly with reabsorption of water and not of solutes.Osmotic diuretics, are filtered from the glomerulus but are not absorbed and tend to remain in the tubular fluid. Hence,in descending limb of loop of henle which is permeable to water, they prevent the movement of water out from tubular lumen since they exert on osmotic pull on water.


 

Biosynthesis of thyroid hormones

Thyroid hormones are iodinated tyrosine which is an amino acid.   Synthesis of thyroid hormones occurs in multiple steps 

 1. Synthesis of thyroglobulin: Thyroglobulin is synthesised by follicular epithelial cells just like any other protein synthesis by cells and is secreted into the colloid by exocytosis. So this protein is stored in the colloid. 

 2. Trapping and transport of iodide: Iodine is present in circulation as iodide ion. This iodide is taken up into the cytoplasm by NaI- symporter which is a secondary active transporter present on the basolateral membrane of thyrocytes. This Iodide then enters into the colloid via the apical membrane through the Cl-/I- exchanger which is also known as ‘pendrin’ 

 3. Organification: So now we got both iodide and thyroglobulin in the colloid. However, the iodide cannot react with tyrosine residues. For that it should be oxidised to iodine. So this process of oxidation of iodide to iodine and iodination of the tyrosine residues of the thyroglobulin is known as organification.  This occurs in presence of the enzyme thyroid peroxidase present on the apical membrane of thyrocytes.  Many tyrosine residues of thyroglobulin are iodinate either at single site forming monoiodotyrosines i.e MIT or two sites forming diiodotyrosines i.e DIT. 

 4. Coupling reaction: MIT and DIT are not thyroid hormones. It is the coupling or combination of these iodinated tyrosines which produces thyroid hormones. One MIT and one DIT couple to form triiodothyronine i.e. T3 and 2 DITS couple to form Tetraiodothyronine i.e T4 or thyroxine in presence of enzyme thyroid peroxidase. This is known as coupling reaction. 

So basically, thyroid hormones form within the thyroglobulin molecule.

When the gland is stimulated by Thyroid stimulating hormone, all these steps of synthesis of thyroid hormones i.e  synthesis of thyroglobulin, uptake of iodide, organification and coupling reaction are stimulated. 

Also, the follicular cells start taking up the stored iodinated thyroglobulin from the colloid by endocytosis. Intracellularly lysosomes fuse with the phagocytosed thyroglobulin and cleave T3 and T4 from thyroglobulin by proteases which are then released from the basolateral side and enter into the circulation. The separated amino acids are reused to produce thyroglobulin. The MIT and DIT which are not coupled are acted upon by the enzyme ‘deiodinase’ which as its name suggests detaches the iodine from the tyrosine both of which are recycled. 

 

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G-protein coupled receptors

G protein coupled receptors have an extracellular binding site to which the hormone binds and an intracellular cytoplasmic tail region to which G-proteins may bind.  

G proteins act as a switch and functionally couple the receptor to their target enzymes or ion channels. Switch means there is an inactive state when the hormone is not bound to the receptor and when the hormone binds, there is activation. G-proteins or heterotrimeric GTP binding proteins is a set of 3 proteins, G alpha, G beta and G- gamma which are present as an inactive complex bound to membrane just near the hormone receptor.   

The alpha subunit of G proteins is bound to GDP in inactive state.When the hormone binds to its receptor, receptor interacts with the G proteins and GDP bound to alpha subunit is replaced by GTP, which is now said to be in active state. This replacement of GDP with GTP causes alpha subunit to dissociate from beta and gamma subunits and move and associate with some other proteins causing  either opening/closing of ion channels or they affect the activity of enzymes like adenyl cyclase and phospholipase C. 

G proteins which stimulate adenyl cyclase activity are known as G stimulatory proteins i.e Galpha s. while that which inhibits their activity ate known as inhibitory G proteins i.e Galphai. The G proteins which affect the activity of phospholipase C are known as G q proteins. The effect of the hormone on the cell depends on the type of the receptor it binds for e.g. same hormone in one cell may have a receptor which is linked to stimulatory G proteins while in other cells it may have a receptor which is linked to inhibitory G proteins. By the change in the activity of these enzymes, second messenger systems or we can say signalling pathways are activated. 

 Adenylcyclase-cAMP pathway 

When adenyl cyclase enzyme is activated, it causes the conversion of ATP to cAMP. cAMP in turn cause the activation of cAMP dependent protein kinase A which causes phosphorylation of some proteins/enzymes in the cell. Now these proteins may be involved in biological reactions or these phosphorylated protein inturn activate or inhibit other proteins which are involved in cellular reactions. So ultimately this process affects the function of the cells. In addition,PKA can also increase/decrease the transcription of some genes.Close to these genes, is present a region known as cAMP-responsive element (CRE) region of DNA.  A subunit of PKA moves to the nucleus and phosphorylates another protein, the cAMP-responsive element binding protein (i,e CREB protein) which then binds to CRE and alters transcription of the genes where CRE is present. The set of proteins activated by cAMP varies in different cells. So adenyl cyclase activation  in different cells leads to different actions. However if same adenyl cyclase is activated by different ligands in the same cell, it leads to same actions. So in this pathway, cAMP acts as a second messenger.

Phospholipase C-IP3-DAG-Calmodulin pathway 

 When phospholipase C is activated by Gq proteins, it causes breakdown of the membrane phospholipid phosphatidylinositol 4,5 bi or di phosphate (PIP2) into IP3, i.e inositol triphosphate and Diacyl glycerol (DAG). Ip3 acts on IP3 receptor (IP3R) present on endoplasmic reticulum. This IP3R is itself a ligand gated calcium channel. When IP3 binds, it opens up causing release of calcium ions from ER into the cytososl. The calcium ions in turn bind with calmodulin causing activation/inhibition of calmodulin dependent protein kinases.  

DAG activates protein kinase C which then phosphorylates other proteins just as we saw for adenyl cyclase dependent kinases. So in this case IP3, DAG and calcium are second messengers.

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Weber Fechner and Steven's Power law

The concept of Just Noticeable Difference was given by Weber where he explained that we will be able to perceive the difference in intensity of the stimulus depending on the original strength of the stimulus. For higher strengths of stimulus, a greater chnage in intensity of stimulus is required is required to notice the difference in intensities. 

Fechner took this concept and tried to explain the percieved intensity of stimulus vis a vis the strength of stimulus. But Fechner law was applicable to only very high and very low intensities of stimulus. Later this was modified by Steven who gave Power law to explain the percieved intensity of stimulus.


Watch the video for details....

Classification of sensory receptors

Afferent neurons transmit action potential from the periphery to the central nervous system. These afferent neurons have sensory receptors at their peripheral endings. Receptors as the term indicates receive information or we can say characteristics of a stimulus from the surroundings. 

The stimuli are of various types depending on the type of energy change for e.g. there can be either mechanical stimulus , chemical stimulus or thermal stimulus etc. Body has receptors for detecting different types of energies. But once the receptors detect the stimulus, it has to be transmitted to the CNS as action potential, so receptors convert the stimulus energy into electrical signals. This is known as signal transduction.  

Sensory Receptors can be classified in 3 ways

1. Based on their location: exteroceptors, interoceptors and proprioceptors

2. Based on modality of stimulus they detect: mechanoreceptors, chemoreceptors, thermoreceptors, nociceptors, and electromagnetic receptors . 

3. Based on rate of adaptation: Slowly adapting receptors and fast adapting receptors.

In skin , their are various mechanoreceptors. However, they are located in various layers of skin e.g. Merke;'s disks are located superficially in dermis and detect superficial texture of the object while Ruffini ending is deeper in dermis and detects pressure. 

Also there are Meissner’s corpuscle versus Pacinian corpuscles. Both these receptors adapt very fast. Thus, they detect vibration sense. But, meissner’s corpuscle respond to slow vibration while pacinican corpuscle respond to fast vibration because pacinian corpuscle adapt very very fast. And pacinican corpuscles are located deep so they respond to deep pressure vibration while Meissner’s has to be superficial like a flutter on the skin. Also, meissner’s corpuscles detect the sensation from a small superficial area while Pacinian corpuscle respond to a stimulus even when it is present in a little larger area. This is known as receptive field of the receptor.

Starling's forces and physiological basis of edema

Starling's forces

1. Capillary hydrostatic pressure

2. Capillary oncotic pressure

3. Interstitial fluid hydrostatic pressure

4. Interstitial fluid oncotic pressure

Forces at arterial end of capillary

Starling's Forces promoting filtration: 

1. Hydrostatic capillary pressure: 32 mmHg 

2. Interstitial oncotic pressure: 8 mmHg (due to proteins)

3. Interstitial hydrostatic pressure: -3 mmHg (Some fluid is also present in interstitial space. However, due to pulling by the tissues, a negative hydrostatic pressure is generated in interstitial space. So, it promotes filtration)

Total =  43 mmHg (by adding all forces)

Forces opposing filtration

2. Oncotic capillary pressure: 28 mmHg

Net driving force at arterial end of capillaries = Forces promoting filtration - Forces oppsong filtration

                               =  43-28 = 15 mmHg

Since it is a positive force, it drives water movement out of the capillaries.

Starling's Forces at venous end of capillary: 

Starling's Forces promoting filtration: 

1. Hydrostatic capillary pressure: 10 mmHg   (hydrostatic pressure decreased due to the movement of water outside the capillaries at the arterial end

2. Interstitial oncotic pressure: 8 mmHg (due to proteins)

3. Interstitial hydrostatic pressure: -3 mmHg 

Total =  21 mmHg (by adding all forces)

Forces opposing filtration

1. Oncotic capillary pressure: 28 mmHg

Net driving force at arterial end of capillaries = Forces promoting filtration - Forces oppsong filtration

                               =  21-28 = -7 mmHg

Since it is a negative force that means it draws in water into the capillaries. So there is the inward pull of water at the venous end. 

That means water which is filtered at the arterial end is reabsorbed at the venous end. 

But at arterial end, the net outward force was 15 mmHg and at the venous end, the net inward force was 7 mmHg so some fluid remains in the interstitial space. Lymphatics carry that excess fluid from the interstitial space. 

Now when can fluid accumulate inside interstitial space? That extra fluid accumulation in interstitial space is known as EDEMA. 

 So when can oedema occur? 

1. Increased hydrostatic pressure occurs in the case of hypertension or excess fluid accumulation. ( heart failure or iatrogenic) 

2. Decreased oncotic pressure that is due to decreased protein concentration. 

It may happen due to:

Malnutrition: Decreased protein intake 

Malabsorption: Decreased protein absorption

Liver disease: Decreased synthesis of proteins

Kidney disease: Increased loss of proteins

3. Increased capillary permeability can occur in case of local injury.

4. Blocked lymphatics can occur in case of filariasis, metastasis of cancerous cells to lymph nodes 

Edema is of 2 types

1. Transudate: This means that it is the water that is being accumulated inside the interstitial space. 

2. Exudate: along with water, there is a movement of proteins also from the capillary.  So exudate is water plus proteins. It occurs when there is increased permeability of capillaries causing movement of proteins also out of the capillaries.