This comprehensive review covers three major areas: (1) Cell signaling mechanisms including autocrine, paracrine, endocrine, and gap junction signaling, with membrane receptors (low ligand concentration, signal amplification, hydrophilic ligands) versus nuclear receptors (high ligand concentration, gene expression, hydrophobic ligands); (2) G-protein coupled receptors with their seven transmembrane helices, heterotrimeric G-protein activation (GDP to GTP exchange via GEF, GTP hydrolysis via GTPase), and G-alpha subunit functions (Gs activates adenylyl cyclase, Gi inhibits it, Gq activates phospholipase C); (3) Molecular techniques including PCR (denaturation, annealing, extension cycles), Southern/Northern/Western blots for DNA/RNA/proteins, and FISH for gene copy detection.
Pre-Midterm MOL Review | Awad Walied | MOL 125Added:
Hello everyone. My name is Wed. I'm a second year med student and today I'll be giving you a very important pal inshallah. Uh today's pal is mal pimemed and we're going to cover the high yield concepts and I've also tried my best to cover everything but I ended up with covering 80 to 90% of everything. If this number is good with you then this PAL is enough for premidterm inshallah.
If not then I recommend you go through the individual pals or the uh lectures itself. This uh I believe 80 to 90% is a good number and for cell signaling I aimed for 95% because cell signaling is easier to cover. Now with that being said um I want you guys to know the format of the entire lecture and it's pretty unique so please focus.
Firstly we're going to cover cell signaling 1 2 and three and it's going to be question based. Now I know this is a recording but I still want you to pause try to actually answer because this is a review. You're expected to know and if you don't get it correct we're still going to go through it.
Okay. And then we have molecular techniques, uh, enzyology and all the Dr. Hana lectures at the end. The remaining three parts are going to be normal teaching and we're going to have a few questions here and there. So stay attentive.
Now let's begin. I just want to show you guys the format of the questions. So I'm going to show you a question. I'll just read the question. You guys should pause and try to answer it. And then I'll explain and then we're going to go back and I'll explain each individual option.
and make sure you understand the question fully.
So let's begin with question one of cell signaling one. Which of the following is correct about endocrine signaling?
3 2 1. Okay, let's begin. So the first form of chemical signaling is autocrine and in autocrine signaling the cell targets itself. Very simple. Auto means self and so it targets itself. The second one is signaling across gap junctions. And over here the cell targets another cell which is connected by gap junctions. And I want you to think of this logically. If it's connected by gap junctions, it means they're literally touching each other.
Okay? So you cannot target a distant cell uh by gap junctions. Doesn't make any sense. And we have one of the options like that. That's why I'm explaining it. The third one is paracrine signaling. And it's when a cell targets a nearby cell. However, they're not necessarily touching, okay, through the ECF or whatever. It's just a nearby cell and it's targeting it. And lastly, we have endocrine signaling and it's when a cell targets a distant cell through the bloodstream. And from the name, you can kind of get that endo means within or inside and this means inside the bloodstream. Okay, it's very important to understand and remember endocrine signaling. Okay, now let's solve the question. Which of the following is correct about endocrine signaling? A. The signaling cell targets itself. We said this is autocrine signaling, not endocrine. B. The signaling cell targets a distant cell through the lymphatic system. We did not mention anything about lymphatic system.
So it's not correct. C. The signaling cell targets a distant cell through the bloodstream.
This is the correct option. and D the signaling cell targets a nearby cell connected by gap junctions. This is not correct. This is signaling across gap junctions. And E, the signaling cell targets a distance cell connected by gap junctions, which does not make any sense. This is how the entire cell signaling part is going to be very simple and very nice. Question two, which of the following is correct about membrane signaling?
Okay, 3 2 1, let's begin. So this is a very nice table which compares membrane receptors to nuclear receptors and the features. Okay. So liant concentration for membrane receptors you have low liant concentration. You just have to memorize it. For nuclear receptors you have high liant concentration and then based on this we can predict the second line. Signal amplification is going to be uh is going to be present for membrane receptors because you have low lian concentration. If you have a low liant concentration, you want to amplify the signal. And if you have a high liant concentration, you don't want to amplify the signal. Okay.
Now, for target cell response, you have multiple target cell responses with membrane receptors. However, with nuclear receptors, you only have gene expression. Very important and high yield.
Now, for the lian type, for membrane receptors, you have hydrophilic lians and hydrophobic lians for nuclear receptors. Um and liant examples you have extracellular lians which include peptide hormones such as insulin, neurotransmitters such as dopamine, cytoines such as interlucans, growth factors such as epidermal growth factor, amino acids such as glutamate and for nuclear receptors you have intracellular lians such as steroid hormones cortisol, thyroid hormones thyroxine, retinoids such as retinoic acid, fatty acid derivatives such as prostaglandon, And finally, amino acids such as thyroxine.
Um, honestly, you just have to memorize it. I can't do much here. So, let's solve the question. Which of the following is correct about membrane signaling?
A, the lian concentration is high is incorrect because we said for membrane signaling the liant concentration is low and then okay, let's look at option B.
Signal amplification is required. This is the correct option.
because we said low liant concentration and then signification is required.
Let's look at it over here. Low liant concentration signification is required.
And option C, the lian type is hydrophobic. We said it's hydrophilic.
Hydrophobic is for nuclear receptors.
The initiation signaling lian is found in the intracellular compartment. Now we said that it's membrane receptors and we said that the lian type is hydrophilic and we also mentioned the examples extracellular lians.
So it's incorrect. This would be correct for nuclear receptors. And finally the steroid hormone cortisol. This should tell you everything is an example of a membrane receptor liant. Incorrect. It's an example of a nuclear receptor liant.
Okay, now question for you guys. One of them is intracellular signaling. The other one is extracellular signaling.
Which is which? I'll give you 3 seconds.
3 2 1. This is extracellular. As we can see over here, the liant binds in the extracellular compartment. Okay. And then it triggers um a signaling cascade. As we can see, first amplification, second amplification, third amplification. So this is membrane receptor extracellular lian extracellular um everything. Okay.
And if if this is extracellular this would be intracellular. As we can see the lian goes through the plasma membrane finds its receptor in the in the cytoplasm and it then binds to a DNA and we can kind of predict from this diagram is that it's affecting gene transcription gene expression. Okay.
Now question three. Which of the following is correct about G- protein coupled receptors?
Again, try to solve it. Then we're going to go through the explanation.
3 2 1.
Oh, there's one thing I forgot to mention, guys, is that the answers of all of the questions are going to be found at the end of the pal. Okay, I have one slide with the answer bank. All of the answers are going to be there. or if you download the notes version of the Canva document, you're going to find the answers under these slides. So, let's explain.
This is one second. This is an example of a G-proin coupled receptor. It's a receptor which is coupled with a G- protein. Okay.
And let's try to dissect the structure bit by bit because it's important to know every single tiny detail. Firstly we have the N terminal in the extracellular compartment outside the cell and over here we can see that the lian binds. So lian binds to the N terminal which is outside the cell. Okay extracellular compartment and then in the C terminal or or C terminus we have the G- protein itself which is the G alpha, G beta and G gamma and this is found in the intracellular compartment. Okay. So, N terminus outside liant C terminus inside G- protein or hetro G- protein.
And we can also see there's three loops outside and three loops inside. These are loops which are strengthened with dulfide bonds. Important to remember three intercellular loops, three extracellular loops. And we have 1 2 3 4 5 6 seven total transmembrane alpha helyses.
So from this you can remember N terminus outside C terminus inside lian to N terminus G- protein to C terminus three loops inside three loops outside both connected by dulvide bonds and seven transmembrane alpha helyses and from this if you can read it I just wanted you to remember that G- proteins are the most abundant membrane receptors this could be a question okay Now let's try to solve this. Which of the following is correct about G- protein coupled receptors? The receptor is found in proarotes and ukarotes. I did not mention this but the receptor is only found in ukarots. Okay. So A is wrong. B. The receptor is made of eight transmembrane alpha helyses. Incorrect we said seven. C. The C terminal domain binds to the lian.
Incorrect. We said the C terminal domains binds to the heterrotime G protein complex. And so C is incorrect.
D the N terminal domain binds to the hetrotic G protein complex. Incorrect.
We said the internal domain binds to the lian. And then E has to be correct. But let's just read it. The receptor has three intracellular loops connected by dulfide bonds which checks out. One more time. Look at the structure. Let's go to question four. And we have eight questions for cell signaling one, four questions for two, and eight questions for three. Okay.
Which of the following is correct about the hetroimeic G- protein complex 3 2 1 Okay. So we have two types of G- proteins. The first one is the monomeic or RAS G- protein and the second one is the large or hetroimemeric G- protein complex.
The first one we can see we have one monomer or one subunit which is the RA subunit and it's bound to a GDP. In the second one we can see we have three subunits G alpha, G beta and G gamma and the G alpha is bound to the GDP. Now from this I want you to remember that RAS resembles a G alpha or the G alpha resembles RAS. Okay. And one thing about them is that they're both GTPases.
So they're going to convert GTP to GDP.
Okay.
Am I missing anything? No, we're good.
Now let's look at what happens when the complex is inactive and it then gets activated the changes. Okay. So over here we have the inactive complex and over here we have the activated complex and this changes the moment we get a lian binding to the is it n terminus or c terminus I know I got this correct it's the n terminus so the lian is going to bind outside this is going to activate the entire complex now the g alpha g beta and g gamma are all going to dissociate from the receptor and then from that the g beta and the G gamma are going to drize and go on their own. Okay. And one thing we can see which is different is that the G alpha is now bound to a GTP not a GDP.
And what happened is we're over here. We were over here in the off state. The GTP is the G alpha bound to a GDP. And when the lian binds um something known as Jeff okay guanine exchange factor comes and it changes the GDP with a GTP. So we can see that now a GTP has a GTP and we removed a GDP and now this is activated. This G alpha with GTP is in the active state. It's going to go and phospholate enzymes. It's going to have its function. Okay. and the G beta and G gamma are drizzed.
They're going to go on their own and do whatever. We don't care. Now, too much activation is wrong. And it's going to again, if you drink too much water, you're going to die. If you don't drink water, you're going to die. Too much from anything is wrong. And the same way we activated the complex, we need to have a way to deactivate or to stop it.
And this is where GAP comes in. GAP stands for GTPs activating factor. and it's gonna activate the GTPs. It's gonna tell it to break down the GTP and chill a bit. So the GTP is going to break uh change into GDP and the phosphate and the complex is going to be in the off state or the inactive state.
Now this was a bit too much quick review. Lan binds we activate the complex G alpha is now with a GTP instead of a GDP. the G beta and G gamma drizz and go on their own. Uh what happens to the GTP in details is that we have GDP with a GTP. GTP is a G alpha and then Jeff changes it to a GTP and then to stop it GAP activates GTPs.
GTPs breaks down GTP and now we end up with GTPs with a GDP. We're back to the start. Okay.
Um let's solve the question now. Which of the following is correct about hetro G- protein complex? A. It is made of G alpha, G beta, and G. Incorrect. It's made of G alpha, G beta, and G gamma, not G.
Excuse me. B. GTP is bound to G alpha only. This is correct. It's not bound to G beta. It's not bound to G gamma. C.
GDP is bound to G beta subunit only.
Incorrect. GDP and GTP whenever they're bound they're always bound to G alpha.
Okay.
Now during GDP inactive configuration G alpha G beta and G gamma are not bound together. Incorrect. We said in the active configuration they are bound together. When the complex is activated they separate. However G beta and G gamma drize and go on their own.
Uh now during GTP the active configuration G alpha and G beta separate from the complex and form a dimer. Incorrect. It's not G alpha and G beta. It's G beta and G gamma. And that's the trick.
Now pretty simple. Let's solve question five. Which of the following G alpha subunits G alpha subunits is responsible for activating adinali cycles?
Okay, 3 2 1 let's proceed. This is the G- protein alpha family and the four of them which you need to know are GQ, GS, GI and G1213.
Now for GI, I want you to ignore this.
Just focus on this. Okay, so we're starting with GQ. GQ activates something known as phospholip C beta and phospholip C beta is going to cleave something known as PIP2. We're going to look into the details which increases D AG and IP3 and we also increase the calcium. Okay, just remember the keywords calcium, IP3, D A phospholip beta, they're all related to GQ.
Um GS is pretty simple. It activates adinalized cycles which increases cyclic AM. Then we have the opposite of that which is GI and GI inhibits adinyl and therefore it's going to decrease the cyclic AMP and finally we have G1213 and G1213 activates roynes and guanulate cycles which increases cyclic GMP.
um you can read on it again pretty simple but we're going to go into the details now let's start talking about G alpha S specifically so over here we have the GPCR pretty simple the G alpha S is found over here gets activated goes on its own and then it activates AC which as we said is addin light cycles AC is then going to change ATP to cyclic AM very important key reaction cyclic AMP is going to go and activate protein kynise A and protein kind A is going to go do its function we don't care about that um what else are we supposed to say okay now as we said too much activation is wrong and it's harmful for us actually so we have to some way or another remove the cyclic AMP and for that we have this enzyme known as phosphodiestase or PDE and it's going to change the cyclic AMP to five prime AMP. So quick revision, G alpha S activates AC, AC converts ATP to cyclic AM. Cyclic AMP activates protein kazine.
And then cyclic AM is converted back to five prime AM to stop the reaction by PD.
And what about G alpha I? G alpha I is going to do the opposite. It's just going to inhibit AC. So if it inhibits AC, we have less ATP being converted to cyclic AMP and we have less cyclic AMP.
So we have less activation of protein kin. Pretty simple, straightforward.
This is the easiest one. G alphas and I.
Now let's talk about G alpha Q. Again, it's simple, but you need to focus. So we have the GPCR over here, G-roin over here. this G alpha Q once it's activated okay actually focus once it's activated it activates PLC beta or PLC phospholip and this PLC is going to go to the membrane find the protein known as PIP2 cut it in half into two parts one part is the IP3 and the other part is the DAG okay now this DAG is going to go and directly activate protein kin the IP3 is going to take a different route. It's going to go to the endopplasmic reticulum.
Um it's going to result in the release of calcium and then this calcium is going to activate the protein can. So if we were to ask what activates protein can directly, it's going to be DAG and calcium, not IP3. Okay?
And then protein can do its function. We don't really care about that.
Now for the last one which is not really G alpha 12 or 13 but this is what the doctor went through because it has a very similar function over here we have something known as soluble guanulate cycles or uh soluble GC okay SGC and what it does is it converts GTP to cyclic GMP this is again if you remember very similar to life cycles however it's for GTP not ATP P. So in the beginning you have nitric oxide. It activates soluble guilate cycles which converts GTP to cyclic GMP. And if we have too much uh then cyclic GMP is going to activate protein kynise G. But let's go back. If you have too much cyclic GMP, phosphodiestase is going to kick in and it's going to convert cyclic GMP to five prime GMP.
Just as we said, phosphosphodest converts cyclic AM to five prime AM in the previous example.
Now let's focus on protein kynesy once it's activated by the cyclic GMP.
Protein kynis is going to have many different functions. It's important you have to read them and memorize them such as smooth motor relaxation, platelet inhibition, and changes in gene expression. Okay, I believe that's it.
Okay, let's go solve the question.
Which of the following G alpha subunits is responsible for activating adenalide cycles? G alpha S, G alpha I, G alpha Q, G alpha 1213, G alpha G alpha X. Okay, the answer is clearly G alpha S. But I want to give you more questions. So we said ads is G alpha. What if it was inhibiting adine?
It's going to be G alpha I. Okay. What if it was uh related to smooth muscle relaxation for example?
This is going to be option D which is G alpha 1213. And finally um G alpha Q is going to be anything about calcium uh PIP2 IP3 DAG whatever.
Okay.
Question six. Which of the following signaling cascades is correct?
Okay, I hope you answered that.
Let's go through the explanation. Oh.
Oh, yeah. You guys should know this already. I did explain it in the previous one. So, let's just answer it directly. Which of the following signal cascades is correct? A. GTP activating protein or GAP converts GDP to GTP. Now, is this correct? No, it's not because we said GAP converts GTP to GDP, not GDP to GTP.
Um, what else? B. AC converts GMP to cyclic GMP. Incorrect. This is a function of GC, not AC.
Um and then it doesn't even convert GMPP converts GTP. Okay.
C uh soluble golate cycles converts ATP to cyclic AMP. Incorrect. That's a function of AC. So A, B and C are all incorrect. Phospholip C or PLC converts IP3 to DAG and PIP2. Again, this is incorrect. Phosphip C converts PIP2 to IP3 and DAG. Okay. So if this comes here it's going to be correct. And then the last one has to be correct which is phosphodiestase converts cyclic AM to five prime AMP. Again checks out.
There's nothing wrong.
Question seven. Which of the following two components are required for enhanced activation of protein kynise C?
We did mention this. You should go all the options are wrong except it's this one. It's D. The correct answer is D. Uh because we said where's is there no DAG and okay there is no DAG and IP3 option. However, if there was a DAG and IP3 option, it would still be incor it would still be incorrect because only calcium and DAG directly activate protein kinosine.
Question eight and the last one for cell signaling one. Which of the following protein kinases is directly responsible for inhibition of platelets?
A3 2 1 The answer is protein kynise G.
And this is it for cell signaling one.
We have four questions only for cell signaling two. So let's go through them quickly. Question one, which of the following receptor tires in kinasis RTKs consists of two chains linked together even before activation?
Okay, you should know this, but we're going to go through it. 3 2 1. TKRS, we have a couple of them over here. And honestly, the only thing you should know is that they drize one when a substrate is bound to them. When a substrate is not bound to them, they're separate.
Okay, there's two exceptions and the doctor likes to ask about them. The first one is insulin receptor. It's always in this drizz form. And the second one is IGF-1 receptor.
Okay, with that knowledge, let's go and solve the question.
Which two chains are linked together?
It's insulin. If insulin was not here, it would be IGF-1. If IGF was present here, okay, question two. Which of the following domains of SRC kynes is responsible for binding to phosphorated tyrosine residues?
Okay. 3 2 1.
Honestly, not much I can do. You just have to memorize this word for word literally because they can ask you about any single tiny detail over here. The first domain over here is the SH1 domain and it's the catalytic domain and it adds a phosphate from ATP. The second one binds to phosphotyroine residues SH2. The third one protein protein interactions with proline rich sequences SH3 and then we have SH4 which is unique the exact function is not known and the N terminal binds to the plasma membrane so let's go back SH1 we said SH1 is the catalytic domain so it's incorrect SH2 is what phosphorated residues So it is correct. It binds to phospho tyrosine residues. The answer is B. And then SH3 protein protein interactions incorrect.
SH4 unique. Incorrect. SH5. We don't have an SH5. If it was the N terminal option, we would say it binds to the plasma membrane. Okay.
Question three. Which of the following proteins is responsible for converting GDP to GTP during RAS signaling?
3 2 1 let's solve.
So what is RAS signaling? This entire thing is RAS signaling and we only need to know a few tiny details. So we said in the beginning that RAS resembles GTP uh is RAS is a GTP resembles G alpha.
Again remember that. Okay.
So the first step over here is we have something known as GRB2 and it binds to the tyrosine kynise receptor in the SH2 domain and then S SOS binds to this GRB2. Okay.
Now SOS is going to go to the RAS the inactivated RAS. So the RAS which is bound to the GDP and it's going to change this GDP to a GTP.
Now recall the similar function which we saw before. So SOS is doing a similar very very sim it's the exact function actually of Jeff the guanine exchange factor.
Now this activated RAS is going to um phosphorolate the raph or promotes phospholation of the raph. Now phosphorated raph is going to do the same for mech. Mech is going to do the same fork and is going to have its function. Okay. So is okay I'm not going to leak the question.
Which of the following proteins is responsible for converting GDP to GTP during grass signaling?
The answer is SOS.
The remaining ones are they have different functions so I don't need to go through them. [snorts] Which of the following proteins is considered the most downstream in the process of signaling cascades? I did explain this over here. So, no more explanations. Let's just solve it. Is it RAS? No, it's not. RAS is one of the higher ones.
Mech. No, Mech is almost there, but the answer is IK if I'm not mistaken.
Yes, it is. The answer is IRK and RAF AKT is it's all incorrect as you can see. GRB2 SOS RAS RAF mech and that is it.
This is cell signaling two. Very quick, very easy. Now for cell signaling three, we have eight questions.
Which of the following is a peptide hormone?
A B CDE E okay 3 2 1 let's explain we have five classes of hormones which you should know about and they are steroids peptide hormones gaseous hormones fatty acid derivatives or amino acid derivatives honestly I recommend memorizing all of the different options but if you really really don't want to then just memorize the first one which is cortisol insulin nitric oxide prostagland abandons and epinephrine.
So which of those are which of those is a peptide hormone? The answer is insulin. Okay, insulin is not here.
[laughter] The answer is B, growth hormone. Okay.
Um what else? So cortisol would not be a peptide hormone. Cortisol would be a a steroid.
And then dopamine from A2 and hydrogen sulfide. Dopamine is an amino acid derivative. Thromboxane A2 is a thromboxane under fatty acid derivatives and um hydrogen sulfide is a gaseous hormone. Question two, which of the following is a gluccocorticoid steroid hormone?
This is ERP knowledge kind of. You should still know it.
So 3 2 1. Let's go through the types of steroid hormones. So we had five different classes of hormones and now we're going to dig deep into this class.
So we have two different direct classifications. The first one is the corticids. Second one is the gonadal hormones. Under the corticids we have gluccocorticoids which is cortisol and mineralcorticoids which is aldoststerone. The way I remember aldoststerone is mineral water and then aldoststerone has this function. Okay.
So if you remember aldoststerone for mineral corticoids immediately just link cortisol with gluccocorticoids. I don't know if I'm making sense. I hope I'm doing.
Now these are the two examples you have to remember only for corticoids.
For gonadal hormones you have male or the androgens and the female sex hormones.
The androgens you have testosterone and DHT. And then for the female sex hormones, you have the estrogens which are estradiol and estrone and the progesterrogens which are progesterone.
With that knowledge, let's solve the question. Which of the following is a gluccocorticoid steroid hormone?
The answer is D. Okay, the answer is cortisol.
Testosterone is an androgen. Estradiol is a estrogen and then aldoststerone is a mineral. or to call progesterone as a pro estrogen whatever that is. Question three, which of the following is correct about a typical steroid receptor structure and function?
Okay. Three, two, one.
Let's dissect the typical story receptor. uh structure.
So in the beginning closest to the end terminus we have the variable domain which interacts with other transcription factors and then we have the DNA binding domain which the key word you should remember is zinc finger motives you don't need to understand it. And then next to that we have the domain for drizzation.
It's where two receptors join. And then we have the hormone binding domain which is the site where the lion binds. And finally the NLS and this is the fifth domain. And the NLS is simply a signal. I think you might remember this from mole one. It's a signal which tells this entire protein to go to the nucleus. Okay.
With that knowledge, let's solve the question. So which of the following is worked about typical storage receptor structure and function? The variable domain is the closest domain to the N terminus. Let's look at that variable domain. First one closest to terminus.
So the answer is A.
Now the DNA binding domain is the closest domain to the C terminus.
Incorrect. DNA binding domain is this domain and it's far away from being the closest to the C terminus.
The drizzation domain is characterized by zinc fingers.
domain for drizzation. It's not the DNA binding domain is characterized by zinc fingers.
And then the NLS is responsible for receptor trafficking to the cytoplasm.
Incorrect. We said it's responsible for uh receptor trafficking to the nucleus.
And then E, the lian binding domain is responsible for receptor drizzation.
Um and this is incorrect because the domain for darization is responsible for that.
Okay.
Question four. Which of the following is correct about a motive?
3 2 1.
Okay. So we have a big table has everything we need to know. Motive and domain. Motives are super secondary in structure. Domains have a tertiary structure. Motives are formed by connected alpha helyses and beta sheets through loops. Domains are formed by dulfide bonds, ionic bonds and hydrogen bonds.
Motives have structural importance.
Domains have functional importance.
Motives perform similar functions to each other. Domains have unique functions.
And finally, motives are not stable independently.
Meanwhile, domains are stable independently.
So, which of the following is correct about a motive? It is such a structure.
Incorrect. This is correct for a domain.
It's formed by dulfide bonds between amino acid chains. Incorrect. This is correct for a domain. Again, it's independently stable. Incorrect. Again, this is correct for a domain.
um it's made of greater than 250 amino acids. Now I did not mention this directly but try to take a guess tertiary structure um dulfide bonds ionic bonds stable independently from this you can kind of get the the the vibe that domains are larger in size compared to motives. So a new fact which we learned is that motives are less than two 250 amino acids mean while domains are more than 250 amino acids. And then finally it primarily exhibits structural stability functions and this is the correct option.
Question five. Which of the following hormone receptors forms a hetrodimer upon lagan binding? And this is where it gets interesting.
3 2 1 Okay, let's explain. So, we have two major classes of steroid receptors.
The first one is steroid membrane receptors, which we're not interested about. The second one is steroid intracellular receptors.
Now, let's divide this class into two.
Type one of steroid intracellular receptors are found in the cytoplasm.
Okay? Everything which is highlighted could be a question. Okay. Uh it's found in the cytoplasm. It's combine combined with HSP heat shock proteins. Type one also forms homodimer from the name homo is same. So it forms DRS with receptors of the same kind. So examples are sexids and corticoids. So corticid receptors are going to form um DR with corticids.
Exactly. it's not going to form a dimer with a sextoid and same for sextoids.
Okay.
Now, type two is kind of the opposite.
It's found in the nucleus. That's the first thing. And it's often bound to the core repressors. Uh type two form hetrodimer mainly with RXR. Even though they do form homodimer, the question is going to be which one forms hetrodimer if there's going to be a question. Okay.
So remember homodimer is type one hetrodimer type two and the hetrodimer is specifically with an RXR receptor.
Homo is same hetro is different. Okay.
So it's going to be either look let's look at the examples.
Okay the examples over here are thyroxine, vitamin A and vitamin D. If we have a vitamin A receptor, it's always going to bind with an RXR receptor. If we have a thyroxine receptor, it's always going to bind with a thy RXR receptor. It doesn't matter what it is, it's going to bind to RXR receptor. Okay.
Now, let's solve the question. Which of the following hormone receptors forms a hetrodimer upon lagan binding?
Is it tes receptor?
No, it's not because testosterone receptors form homodimer. They're type one. Cytoxin receptors is the correct option. Uh they form their type two receptors and then estrogen receptors type one progesterone receptors type one gluccocorticoid receptors type one.
Let's move on to question six. Which of the following receptors is associated with the longest hours today's signal response time?
I'll show the answer in 3 2 1.
So pretty easy. You just have to memorize it. Moving from slowest to fastest, we're first going to find the steroid receptors. An example is nucleus initiated steroid signaling. You also have membrane initi signaling. This is not important. So steroid receptors slowest hours to days.
And then you have GPCRs, TKRS, nonTKRS, and Jackat receptors. They're all seconds to minutes. And the fastest fastest ones are the ion channel receptors and their speed is signal response time is milliseconds.
So if we were to solve this, the longest is going to be the steroid receptor. The shortest is going to be the ion channel receptor and in between we're going to have B and C. We didn't mention anything about cytoine receptors.
So the answer here is D.
Question seven. Which of the following is correct during aldoststerone signaling? You guys should know the answer for this because we explained it on already.
So aldoststerone binds to an aldoststerone receptor on the plasma membrane. Incorrect. Aldoststerone is type one. So it's going to bind in the cytoplasm.
Um, okay. This kind of gave the answer.
Aldoststerone binds to an aldoststerone receptor in the cytoplasm. So option B is correct.
And then aldoststerone binds to an aldoststerone receptor in inside the nucleus. Incorrect. It would be correct if aldostrone was a type two receptor.
However, aldoststerone is type one.
Okay. Aldostrone receptors form hetrodimer with RXR receptors.
Incorrect. That's for type two. And then lastly, aldostrone receptor binding to heat shock proteins promotes transllocation to the nucleus. Now even though aldostrone receptors are bound to heat shock proteins as we said before this doesn't happen the promotion of transllocation to the nucleus is incorrect. Heat shock proteins actually prevent that. So when we get an aldoststerone molecule it binds to the aldost receptor it moves the heat shock protein away and when the heat shock protein away the heat shock protein leaves then we uh there's transllocation to the nucleus.
So it's quite the opposite and that's why E is wrong.
The answer here is B. Now question eight.
Which of the following is correct during vitamin D signaling?
3 2 1. Okay. First of all, we have to understand vitamin D receptors are type two receptors.
So uh they bind to vitamin D binds to vitamin D receptor on the plasma membrane. incorrect uh in the cytoplasm incorrect um and then inside the nucleus correct so the correct option is C now let's look at those D and E vitamin D receptors form hetrodimer we're almost correct we're correct until now and then with R receptors it's incorrect we said always with RXR and then option E says vitamin D receptors bind to heat shock proteins in the cytoplasm to promote lian receptor transllocation to the nucleus.
Incorrect. We don't bind to Hock proteins, anything. It does bind to codepressors though.
This is it for cell signaling 123. Take a quick break and be refreshed for molecular techniques.
Okay, we're back. So, let's begin with molecular techniques. one texture 4 and honestly very easy. Uh okay before we begin I just wanted to say this is a very very very compressed um review on molecular techniques. If you want the full package I recommend watching my previous pal which is the entire molecular techniques thing in 1 hour and 10 minutes. uh should be good should be inclusive inshallah and you should focus on postmidterm more than premidterm so don't go and start studying the slides from the beginning all of that I don't think you have time for that now let's actually begin again DNA analysis you have three core pillars resection the nucleases cloning of DNA and probes what are section nucleases they are enzymes which cleave DNA into smaller more manageable fragments and they're used for paternity testing thing something about them is that uh they're very specific to restriction sites and most of those restriction sites are palendromes.
Now cloning of DNA uh this is basically where we amplify DNA by inserting this DNA uh fragment into a plasmid and then we insert this plasmid back into a bacterial cell. the bacterial cell is going to replicate and the plasmid is going to duplicate with the bacterium and since we duplicate the plasmid we're technically duplicating the DNA itself and then we just collect those plasmids and extract the DNA from it okay and finally we have probes uh probes are pretty pretty easy we explained them a lot and they basically bind to DNA RNA or proteins and they give off a signal This is the highlight of a probe. It gives off a signal. And we have different types of probes. Double stranded DNA, single stranded DNA, and RNA and antibodies. They're all probes or can be probes.
Now, this is important and I think you're going to get a question on this, hence the star.
uh fish the fish technique it's a technique which detects the number of copies of a gene in a cell using cDNA probes uh what I mean by this is that if you have amplification of genes or deletion of genes this is going to be detected using the fish technique um again we mentioned previously that uh fish is used to detect for her two positive breast cancer and over here we a breast cell. Okay, we don't know if it's cancerous or not. We use the fish technique and we visualize the number of um genes present over here. Okay, and it specifically detects the epidermal growth factor too. Normally, you're supposed to have two reds and two greens. If you have more than that, there is gene amplification. If you have less than that, you have gene deletion.
And with her two positive breast cancer, the breast cancer cells tend to amplify the her two gene and therefore we can see it a lot. Over here we have seven uh red dots which means we have seven uh genes her two genes present. Okay. All you need to know is um in a normal cell you have two reds and two greens. More than that is gene amplification. Less than that is gene deletion. And with her two passive breast cancer cells there is gene amplification.
Now this is low yield but I would still recommend if you review it dot bloods there they use al specific olon nucleotides and gel electropheresis you got questioned on this so much in the midterm I don't think you're going to get questioned in the final but even then if you have time go and review it.
Now for molecular techniques question I think we have one question only.
When using fish technique to evaluate breast cancer a breast cancer sample which finding what finding in a cell would indicate that it is a normal cell rather than a heru positive breast cancer cell 3 to 1. Um the answer is going to be C because in a normal cell you're going to find two red signals and two green signals signals. So a the presence of a gene deletion with only no the amplification of the her two gene shown by seven no that's for her two positive not a normal cell and then the use of a cDNA probe to detect an altered number of gene copies there is no altered number of gene copies because it's a normal cell.
Now for micro techniques 2 I think this is the only slide we have for micro techniques 2. It's so high yield. You're definitely going to get a question on this. So transfer analysis techniques just memorize this pneumonic and copy these and paste it into here. You're not going to get anything out of these. So we use southern blotss for DNA and some examples of that is RFLPS and VNTRS.
They're all related with DNA. RFLPS is the restriction fragment length polymorphisms.
Uh we're going to look into it, I think.
and the NTRS are the repeats. They're all DNA.
They're all related to DNA. And if you see them linked to any of the blots is going to be southern blot. [snorts] Now we have northern blot after that and it's uh RNA.
Western blotss used for proteins. So again, southern blots used for DNA.
Northern blotss used for RNA. Western blotss used for proteins. Snow drop.
[clears throat] Write the words on top of snow on top of drop and then match it. S with D means southern with DNA. N with R, northern with RNA, cancel the O's, W with P, Western with protein.
Now, let's test your knowledge on this.
They're not going to tell you DNA.
They're going to be sneaky about it.
They're going to say something about, for example, if they say a transcript.
First of all, you're going to think of having the DNA. Then transcription happens. You then have mRNA. So, it's going to be an RNA. The answer should be northern block. What if they say after translation? After translation, you have a protein. So, the answer is going to be western block. And what if they say an enzyme?
The answer is going to be western blood again cuz enzymes are proteins.
Now, let's solve a question on this.
Which of the following laboratory techniques is correctly matched with the macroolelecule it's designed to detect and analyze?
3 to one. The answer is okay. Let's go through them. A northern blot with DNA.
Incorrect. Uh northern blot is with RNA.
Western blot with RNA. Incorrect.
Western blot is with proteins. Southern blot with protein. Incorrect. Southern blot is with DNA. And northern blot with RNA is the correct option.
Now for molecular techniques 3 applications of VNTRS. Okay, so we're not going to talk about RFLP. So we're going to talk about VNTRS. [snorts] Anyways, um VNTRS uh stands for variable number tandem repeats and I think they're going to question you on the clinical aspect of it this time. So let's look at what happens actually. Um normally you'd get a copy of the gene. you'd get um a chromosome from your mother, chromosome from your father, and therefore you're going to have two different copies of genes.
And therefore, you're going to have two different numbers of VNTRS. One which matches your mother, one which matches your father. And if you ma if you sequence your g uh your genes, you're going to find that if your parents are actually your parents, you're going to find that the mother matches partially with the child and the father matches partially with the child. This is correct matching. Okay, now let's look at this one. Um the mother matches the child, the child matches with the father. It's correct. If they don't, then something is wrong. And they're also used for DNA fingerprinting because we all have unique number of VNTRs and a unique sequence of VNTRS. Um they can basically take some DNA from the crime scene and then they can match it the VNTRS with the suspects and we can clearly see that suspect 2 matches the crime scene DNA and this is an application of VNTRS.
Now let's talk about PCR. PCR stands for polymerase chain reaction. It's a series of reactions that amplify specific DNA sequence.
And the first reaction is dennaturation which is at a very high temperature followed by annealing which is at a relatively lower temperature and primers anal in this step. And then we have extending which is in a medium temperature 70 to 75. It's still high but relatively it's medium.
And TAC polymerase extends primers in this step. That's why it's known as extending and tac polymerase is a heat resistant DNA polymerase.
We'll meet here again. So okay, there are a couple things which are needed for PCR to actually um start.
The first thing is the DNA which you're trying to copy.
The second one is primers. And don't worry about the diesel. I don't think they're going to ask you. just read them on your own. Uh the second one is primers and then we have TAC polymerase which is the heat resistant DNA polymerase and finally we have the DNTP mix which is the building blocks the A the G the C the T. Okay.
Now we can see over here that with PCR the DNA strands increase exponentially every cycle. So if you start with two, you're going to have four at the end of second cycle, four at the end of second cycle, and then 8 16 32. Okay. [snorts] Um that's an idea you should just get from PCR. Okay. And then there's a couple of types of PCR.
The first one is conventional or endpoint PCR and it's qualitative. It tells you if DNA is present or DNA is not present. An example of this is we're going to go through it in a bit. This one, this is conventional PCR. It either tells you if the DNA is present or if it's not present. It doesn't give you a value.
Um, and then realtime PCR is different.
It's uh stands for realtime PCR, also known as QPCR. Okay, quantitative PCR.
And quantitative, it tells you the number of copies. It gives you a relative or the actual number of copies.
And this is the more advanced type of PCR. And it has probes that generates fluoresence.
Um, these are some precautions for PCR. Um, just read through them. And some important keywords. This, the way I'm thinking of your final, it's going to be more clinical, more application based.
And so they might actually question you on this. Some key words if you find them the answer is immediately PCR the prenatal diagnosis pre-implantation genetic diagnosis IVF corionic villis and a note RTPCR stands for reverse transcription PCR this has to go this way for okay RTPCR is reverse transcription PCR and does not stand for realtime PCR RTPCR you use um RNA and you convert it back to DNA Yeah.
Now let's look at this example of conventional PCR. [snorts] Um reagent blank. You're not supposed to see anything. And then this is a sample which is known to be negative for EBV.
EBV is Epstein bar virus. We're testing specimens for Epstein bar virus. Okay.
And then this is a sample known to be positive. So until now we know that the test is accurate. And then we have two samples from specimen one. they both test positive for ABV. Two samples for specimen two, they both test positive for ABV. So from this we can say that specimen one has EBV. Okay, but it doesn't end here. We do another test with a protein known as betaactin and betaactin is supposed to be found in every single cell of the body. So if the sample does not show betaactin, it means we don't have a proper sample. Okay, now let's look at the results. With a negative sample, of course, you're not going to see betaactin. Positive sample, you're going to see betaactin. Specimen one and specimen 2. Specimen one showed tiny tiny tiny bit of betaactin. And specimen 2 did not show beta actin at all. So this clearly shows us that the sample has not been collected properly and these results are not accurate.
Okay.
Okay, let's move to realtime PCR or QPCR and they have two different probes which you should know about. The first one is Cyber Green, excuse me. The first one is Cyber Green which is DNA based and then we have Tacman which is cleavage based. And what this means is very simple actually. Over here we can see that the DNA is forming. This is the annealing step uh denaturation I mean and the DNA are basically coming closer to each other analing and then the moment the primer binds you can see that the cyber green dye or the primer or the probe I'm sorry binds to the double stranded DNA and then it gives us a light signal. So we can basically detect the signal and know the amount of double stranded DNA which is being formed and from this we can predict the number of um double stranded DNAs we have. Okay.
So we're going to have in the analing step when the primers bind we're going to have some little signal because technically it's still double stranded DNA and then in the extension step when we actually have double stranded DNA forming we're going to have way more uh cyber green dye giving us a fluoresence and we can detect that. Now for Tacmen which is cleavage based the Tacman probe is going to bind to the single stranded DNA in the beginning and then as the DNA strand is growing it's going to reach this probe it's going to cleave it and at the two different ends of the probe you have R and Q. R is a reporter and Q is a quencher. What R is it basically gives off a light signal. Okay, so R is shining and then Q is absorbing all of that. So we cannot detect it. But the moment they're separated, Q goes away and R goes away. R can actually it's like they're giving us a lesson. R can actually shine. It can give off the fluoresence and we can detect this fluoresence. Okay. So if the if the probe has been broken, it means double stranded DNA has been formed and we can detect this fluoresence from R. Okay.
Okay, now let's talk about real time PCR analysis. And the graph may look confusing. It's pretty simple though.
The first phase, this horizontal line is the lag phase. Okay. And even though we have uh increasing copies, the machine cannot detect it. The fluoresence is too low for the machine to detect.
Now after that we reach the log phase which as you can see the graph is going up and over here we have exponential growth and the machine detects it and then after that we have the plateau and the reason is unknown don't worry about it and one more thing I want to mention over here is the CT or threshold cycle and threshold cycle simply means the number of cycles needed to reach the threshold and memorize it if we don't understand it. But the higher the number of DNA copies we start with, the lower the number of cycles we need and vice versa.
Okay, let's move on to the low yield concepts. Uh they're low yield. I'm not going to mention them, but if you have time, review them. The first one is Sanger sequencing. You got questioned a lot in the midterm. I doubt you're going to get some questions in the final. And then microarrays, they're not part of your curriculum anymore. I don't they shouldn't be reviewed actually. Um incorrect heading. But uh I wanted to address this confusion because a lot of people asked me about my slides last time. The slides were prepared um before your last lecture and so I had to include microarrays just to be safe. But the doctor said you're you're not required to know microarrays. So don't study microarrays at all and then COVID 19 tests they're low yield but if you have time review them now question one couple undergoing IVF ops for pre-implantation genetic diagnosis to screen embryos for a specific just you have to know it already okay without reading the question for a specific single gene disorder before uterine implantation which of the following laboratory techniques is most commonly utilized to rapidly amplify and detect the targeted gene sequence from a single biopsy embriionic cell.
The answer is B. PCR. Don't you just have to memorize this. Okay.
And we're done with six lectures. Six more to go. Um we're at the halfway mark. I recommend you take a break and like 5 minute break. See you after that.
Okay, let's continue. So, how do enzymes work?
Enzymes are basically proteins which reduce the free energy of activation needed for action to take place. And this is the free energy of activation. So the purple line is with the enzyme and the blue line is without the enzyme. If you're color blind, this line is without enzyme uh with the enzyme I'm sorry and this line is without the enzyme. And as we can see the only difference the enzyme does is it reduces this energy needed for the reactants to reach this line or this line. And this line is or this state the peak state is known as a transition state. Okay.
So quick recap. Enzymes are used to reduce the free energy of activation needed for a reaction. And the free energy of activation is the energy which takes the reactant from their starting energy to the peak which is the transition state.
Okay?
And after that they're all going to undergo the reaction with an enzyme or without an enzyme and they're going to become products. The difference in energies between the reactants and the products is known as the free energy of the overall reaction. And very important uh point to remember is that if you use an enzyme or if you don't use an enzyme, if you actually end up moving from the reactants to the products, the free energy of overreaction remains unchanged. There's no change to it.
Simply it's products energy minus reactants energy. No change. So there's no effect on the over I'm just waiting for this to move. Uh read the bottom line. There's no effect on overall reaction energy change. It's also known as Gibbs free energy. So Gibbs free energy remains unchanged.
Now there are six major classes of enzymes and they have different reactions which they catalyze.
The first one is oxidor reductases and from the name you can think of oxidation and reduction reactions or redux reactions if you know them. And we can see that A plus NAD+ give us a B plus NADH. Just remember oxidation reduction is oxidor reductases.
Now reaction two is transferases and from the name transfer. So you're going to have A and B. You're going to transfer one part to uh the other and one part to the other. This is going to give us two new compounds which are C and D. So A plus B give gives us C plus D. And then we have lies and lies. lies comes from the word liate which is joining together. So we're going to have a and b join them together they're going to give us c and li is the opposite.
You're going to start with a com uh A for example. You're going to break it apart giving us B and C.
And then we have hydrolaces from the name hydro is water. And then the remaining part just remember water and water is used to cut something.
Okay. Hydraise. So water is used to cut compound A. It's going to give us compound B and compound C. So a plus h2 gives us b plus c. And finally isomearray um I'll use this enderman plus sheet to help me explain. So isomers are basically they they have the same number of atoms and same number of bonds and okay not necessarily same number of bonds same number of atoms but they're just arranged differently. So if we start with a you're going to end up with a different a like a flipped a or whatever it's going to be b. So, if we have this enderman plushy over here and we take its head off and attach it to its side, it's still going to be like the same number of atoms totally, but it's going to be a different look and therefore they're going to be isomers. So, we're going to start with one compound. We're going to end with one compound. However, they're different. No new atoms added to the entire thing. Now, this is important and you may get a question on this on any of those. Remember quick review oxidation reduction oxid oxidase transfer transferases cut like uh uh I mean join lies cut lies cut with water is hydraise and change the looks or shape as isomease and then we have zyogens and zyogens are the inactive precursor of an enzyme and they usually end with the suffix gen. So an example is pepsinogen which gives us pepsin.
Now let's look at what affects enzyme activity. We have two important factors.
Ignore the third one their temperature and pH.
And one thing about pH uh which okay before that let's talk about how they affect enzyme activity. If you have a very high temperature enzymes will denature and they're not going to work anymore. If you have very extreme pH either very low or very high enzymes will also denature.
If you have a very low temperature the enzyme will be inactive but it will not denature.
Now let's talk talk about Q10 which is the temperature coefficient and it simp it simply stands for the multiple by which the reaction rate increases when we increase the temperature by 10°. So let's look over here at 30 the enzyme activity or the reaction rate was over here. If we increase the temperature by 10° and take this recording, we can see that it's eight. So the first one was four, this one was eight, it doubled and so the Q10 or the temperature coefficient is two cuz it's the multiple. Okay. [snorts] Um and then let's look at this um enzyme activity over pH graph. And we can see two different enzymes. They have two different optimum pH values and they denature when the pH values are extremely away from their optimum values. Okay, now this is an over complicated concept for some reason. I don't know why. Very simple. Just focus with me. Firstly, we have a part of an enzyme, the protein portion. Okay? And it's inactive of course because it's only a part and this is known as an apple enzyme. Okay. So protein portion of the enzyme inactive is the appleo enzyme. Then we have the co-enzyme or the co-actor and it's the nonproin portion of the enzyme also inactive.
And then they come together and they form this holo enzyme which is protein plus non-proin portions. It's the whole enzyme and it's active. Okay, very simple. You start with the protein part, apple enzyme, the non-proin part, the co-actor or the co-enzyme. It binds and then now you have the holo enzyme and then after that you can add the substrate and whatever.
Now let's look at different types of holo enzymes. The first one is appleen enzyme with a co-enzyme and an example of co-enzymes are specifically organic molecules. Okay. An example of a co-enzyme which is an organic molecule is NAD+.
Um and then we have prosthetic groups which are tightly bound co-enzymes. So you're going to have the apple enzyme bound tightly with a prosthetic group and the prosthetic groups are co-enzymes. However, they're tightly bound and an example of this is FAD+.
And then we have apple enzymes bound to metal co-enzymes.
And the metal co-enzymes are known as co-actors. Okay, three different types of holo enzymes.
Pretty simple. Now, let's talk about isoenzymes.
Um, in one line, they're the same. They they have the same enzyme activity, but they have different amino acids sequences and they're coded by different genes. Remember this same enzyme activity but different amino acid sequence coded by two different genes.
[snorts] Uh now two graphs I want you to remember the shape of. Don't understand just remember it. The substrate concentration over the reaction velocity. When you don't have an enzyme and the reaction is taking place, this is going to be the the shape of the curve. It's going to be linear. But when you have an enzyme, it's going to be a hyperbolic shape.
Okay? You're going to have this exponential increase and then slowly it's going to plateau where it reaches the VMX. [snorts] Now, question one. Specific biochemical reaction involves the rearrangement of atoms within a single substrate molecule. converting it into a structure or isomer without adding or removing any atoms. Which class of enzymes is responsible for catalyzing this process?
[snorts] I'll give you 3 seconds. 3 2 1. Is it a isomerase? Yes, it is. Uh as we explained using this guy, um this is exactly what isomeraes do. Question two.
What is the effect on the overall reaction energy or gibs free energy after enzymes are used?
3 to one. The answer is no change. As we said, Gibbs free energy does not change after enzymes are used, before enzymes are used, with without enzymes. It always doesn't change.
Now, let's move on to lecture eight.
Enzyme kinetics.
uh we have two important hallmarks which are vax and km. Vmax is the maximum velocity and then we also have v 0 which is the initial velocity. It's going to be over here and then km is the substrate concentration as we can see substrate graph substrate concentration at half of the vmax. So you're going to get the vmax divided in half and then if you find the substitute concentration it's going to be km or vice versa. Okay?
And km is important because it's an inverse measure of affinity. If you think about it, if the km if you need more substrate concentration or the km was at a higher substrate concentration to reach the same vax, it means the enzyme and the substrate don't like each other that much. And so the higher the Km the lower the affinity and the lower the Km the higher the affinity. Just memorize this. Okay. [snorts] Now using this KM equals substration and at half Vmax they're going to create this equation. Okay? You don't have to create it on your own. You just have to memorize this format. And the reason they do this is because they want to include everything in a straight line in a linear graph because when it's a linear graph you can take the y intercept and the x intercept and they're going to give you the km values if you try hard enough. Okay. So from this we can see that if you read it the intercept the y intercept the place where the graph crosses the y-axis is equal to 1 over vmax. So whatever value you get over here let's say you got two you're going to do 1 / 2 which is going to give you 0.5 and this is going to be the vmax value.
Let's say you got this value and this value is it's to the left of the y-axis.
So it's going to be negative. Okay? So 0 1 2 3 to the right. Then 0 - 1 -2 - 3 to the left. So let's say you got minus3 over here. You're first going to multiply it by -1 - 1. Get rid of the negative. And then you're going to do 1 divided by this value which you got over here.
This is going to give us 1 over 3. And therefore the km is 1 over three.
Um you just have to memorize hon.
And then there's a couple different types of feedback which you should know.
Um the first one is negative feedback or positive feedback. And this is basically when a product of a reaction comes back and tries to slow down the reaction.
This is going to be negative feedback.
Okay. Positive feedback would be if it goes back and it makes a reaction faster. Okay.
Now, if we take this negative or positive feedback and we couple it to another reaction, it's going to give us a different type of feedback. So, let's look at this. A is converted to B, B is converted to C, C to D, D to E. And then when E is converted to CO2 and H2O, ADP is also converted to ATP.
Now this ADP is going to go and give a positive feedback to this enzyme.
This ATP is going to go and give a negative feedback to this enzyme. Okay.
Then finally we have forward feedback in which the substrate itself influences the enzyme to increase its rate of reaction or whatever. Okay. It's pretty low yield this one.
Now let's talk about alossteric enzymes.
Alceric enzymes are enzymes that are regulated by the products of the pathway they control. Basically, they control this pathway. They convert A to B. One of the products in this pathway is going to control it. Remember this. Okay. One of the products in this pathway is going to control it. So, it thinks it's in control, but it's technically being controlled. Okay. [snorts] Um and one more thing about alossteric enzymes is they also catalyze the committed step. Uh you should remember this from mole one. Committed step rate limiting step same thing. And they have different curves. Um when we plot the substrate concentration and the reaction velocity the green one is the aloseric enzyme and it shows a sigmoid curve. The blue one is the miklis mentin enzymes and they're not controlled by products of the reaction and they show the hyperbolic curve which we saw before.
Now we have a couple of enzyme inhibitors. Irreversible class and then reversible. The reversible class is subdivided into three. Competitive where the inhibitors bind to the active site.
non-competitive where the inhibitors bind to the enzyme but not the active site or they bind to the substrate and then you have uncompetitive where the inhibi inhibitors wait for the uh substrate to bind to the enzyme and then they hold it all together. So this is the enzyme and then substrate comes in and then the inhibitor is going to come and hold all of them together. So the substrate cannot leave and this is uncompetitive inhibition.
Um and then these are the different graphs of them. You should memorize all of them and know why they look different. Okay. So for competitive inhibition we can see that the x intercept is closer to the zero and so um the closer it is the higher the km and the lower the affinity because if it's closer it means the value of 1 / km is going to be less and so the value of km is going to be more.
Um, if I'm not making any sense, I have a summary table for you to memorize, okay? But it's pretty simple. And the vmax, the k uh the vmax is not changing at all because the x the y intercept is the same.
And then over here, the km is not changing because the x intercept is the same, but the vmax is changing. The vmax, the y intercept is higher in this example. And so the vmax is going to be lower. It's always the opposite.
KM is uh the X intercept is smaller. KM is going to be higher. Uh the Y intercept is higher. Vmax is going to be smaller. [snorts] And then with uncompetitive inhibition, you have uh a smaller Vmax and a smaller KM.
And the only one which um the only type of inhibition from those which is improved with increasing the substrate concentration is the competitive inhibition.
Uh this is a summary table. Let's go through it quickly. [snorts] With competitive inhibition the bindings to the active site. With non-competitive inhibition the binding is to the enzyme worthy substrate. And with uncompetitive the bindings to the enzyme substrate complex. [snorts] With competitive inhibition the Vmax is unchanged. the KM increases.
Non-competitive the Vmax decreases and the KM is unchanged. And with uncompetitive the Vmax decreases and the KM decreases. Both of them decrease.
And then uh with competitive inhibition only uh it can be overcome inc by increasing the substrate concentration.
Now let's solve this question. Which type of inhibition is shown in this graph?
3 2 1. Okay, let's interpret the graph first. We can see that the km did not change but the vmax decreased because the y intercept is higher now. So the answer is B. Non-competitive inhibition.
Now for the last enzyology lecture, clinical enzyology.
We're almost done, guys.
So we have three different clinical applications of enzymes. The first one is diagnostic. Second one is therapeutic and laboratory. Don't worry about therapeutic. For laboratory just remember Eliza.
And then for diagnostic um we're going to talk a lot about this.
The entire lecture is basically about diagnostic applications of enzymes. And they focus on plasma enzymes. So they measure the plasma enzymes levels. If they're less than normal, then this is an inborn error of metabolism. It just means you cannot produce this enzyme.
If the level of the plasma enzyme is more than normal, it could either mean there is cell damage, so the cells are damaged and they're leaking this enzymes into the blood, or there's cell proliferation.
And we're going to focus on different examples of cell damage.
The first enzyme which we're going to the enzyme class which we're going to talk about today is creatine kynes and we have three iso enzymes of creating kynas. First one is CK1 or CKBB and it's elevated in brain or CNS diseases waterbick.
Okay. So CK1 or CKBB is elevated in brain or CNS diseases. CK2 or CKMBB is elevated in acute MI or micardial inffections. So heart. Okay. Then CK3 or CKMM is elevated in muscle damage. All of these are important. You have to link it together. Okay. So if I ask you which one is elevated in um heart diseases or in acute MI, it's going to be CK2 or CKB. You should also know both names.
[snorts] Now let's talk about the LDHS enzymes.
We spoke about the CK is enzymes. Let's talk about the LDH isoenzymes. Now the total LDH value uh is nonspecific because each LDH isoenzyme points towards a specific marker. Okay? So LDH1 is cardiac specific. So we're going to see a rise in MI for example. LDH2, LDH3, don't worry about them. LDH4 and LDH5 are liver specific. So you're going to see an increase in a liver disease for example. Now application for you guys. This if this is normal normal LDH1 normal LDH2 normal LH3 4 5 what would this be?
Think about it.
So let's compare the LDH5 43 543. They appear to be normal relatively. LDH2 to LDH3 relatively the same. LDH1 is more than LDH2 clearly indicating LDH1 is increased.
And so LDH1 cardiac specific MI. These are how the questions are going to be.
Now let's talk about cardiac markers.
Very very very high yield. Very high yield. For cardiac markers you have three main cardiac markers which are used nowadays. myoglobin, troponin I and CKMBB. CKMBB is also CK2.
Now, troponin I is the most specific cardiac marker. This is a question guaranteed. Most specific cardiac marker troponent eye. And let's talk about the values over here because they're going to like point us in the direction of when to use myoglobin, when to use troponent I, when to use CK and B.
Myoglobin is used in early diagnosis because it's the first cardiac marker to increase. Choponin I is used for late diagnosis because it goes back to normal values after the longest time. And just remember CK2 or CKMBB for recurrent MIS. Okay.
Now, as for the values, I hate to say it, but you have to memorize them. You could get a question on this. What happens? Um, if we're like 3 hours, 3 hours is going to be myoglobin. If it's more than 3 hours, then you could use any of them, you know. If it's more than 4 hours, you could use any of them. And then if they mention anything about a few days later, uh, 6 days or whatever, 4 days for example, it's going to be troponent eye because used for late diagnosis. So, you have to memorize the values. Quick recap. Myoglobin early diagnosis troponent [clears throat] I late diagnosis CK2 or CKMBB recurrent MI.
Question 10. [snorts] Which biomarker is expected to be elevated in an individual who experienced a recurrent heart attack within 6 days?
Okay. So keyword recurrent the answer is going to be CKMBB. Okay. Again CKB recurrent MI troponin late diagnosis myoglobin early diagnosis.
What is the main reason that troponin is preferred over creating kynise in the diagnosis of MI?
3 2 1 this one you just kind of have to remember it gets repeated a lot as well.
Um the answer is E. Troponin is more specific than creating kynes. As we said before troponin I is the most specific cardiac marker.
And lastly we have this very very important enzyme table and I believe two three questions from this. So the first enzyme is acid phosphotase and the source is the prostate only. So the only uh diseases in which you're going to see a rise in acid phosphotase is going to be prostate related which are prostate cancer and prostatitis inflammation of the prostate. Um I think this is going to be a question because it's newly added to your slides. And then you have alpha amiles which uh is produced by the pancreas and the salivorary glands and therefore the diseases are going to be acute pancreatitis and mumps. Then you have lipes which is only produced by the pancreas and it's going to be increased in acute pancreatitis. Now let's say they give you the a question says you have an increased value of alpha amiles and you want to determine if it's a pancreatic issue or it's lever gland issue. What other test result could you order?
The answer is going to be lipase because if you get the lipase uh test results and it's not increased it means it's not a pancreatic issue it's a salivary gland. However, if lip pace is increased, it's going to be a pancreatic issue. Okay.
Now, let's move on to gamma glutami transpidase GGT and the source is the liver and therefore it's elevated in liver diseases. And then we have ALP.
ALP the source is the liver and the bone and therefore it's elevated in liver and bone diseases. Just like we explained this alpha and lipase example, a question could come in GGGT and ALP.
And then we have ALT and A. You guys know them a lot by now. It came in the midterm, I think. And ALT is produced in the liver. A is produced in the liver and the heart. And the important thing you should know for them is that if the ALT value is increased more than the AS value, this is indication of viral hepatitis. However, if A is more than ALT, it's alcoholic liver disease. And the way I remember it is AL. The L reminds me of alcoholic, but it's quite the opposite. So, if AS is more than ALT, it's alcoholic. Then the other one is viral hepatitis.
Um, I'm sorry if I confuse you with this one.
Question three. A woman presents to your office with elevated ALP levels and bone issues. Which additional test should you consider? So remember the options elevated ALP levels and bone issues. ALP measures for liver and bone.
So you want a test which is going to tell us is it a liver only or a bone only. Let's go back. Do we have any other bone only? No, we don't. So we need to find a liver only test. It's either ALT or GGT.
And the answer is C. GGGT. Okay, pretty straightforward, but you have to remember each individual enzyme and the sources.
And with that, we're done with the enzyology lectures. Um, one last push. I know you guys are burnt out already, but let's finish the last three lectures.
They're very very simple and we can finish in a few minutes in trouble. So insulin is as you guys should know by now insulin is the anabolic hormone and it's the hormone which is increased in the fat state. So by anabolic I simply mean it builds um you know they say anabolic steroids whatever it builds muscle.
Okay. So insulin is going to always be building. It's going to build build build build and the source of insulin is the beta cells in the pancreas. The trigger for insulin's release is increase in blood glucose, increase in amino acids and increase in encritins. Encrretins such as GLP1 and GIP and the effects as we said anabolic effects which are building or storing.
And then uh something which is unique about insulin is that it's produced in equimar amounts with something known as ceptide.
The ceptide is a part of the insulin precursor. So you're going to have pro-inssulin and then okay let's say the structure is actually somewhere like this. So you have pro-inssulin it's cut insulin goes in the body and ceptide also goes in the body. Okay. So they're produced in equal amounts and the way the the importance of ceptide is because it has a longer halflife and it's an indication of indogenous insulin. So I'll give you this scenario.
Let's say we have a diabetic patient and we inject him with insulin. Okay? We want to measure the amount of insulin his body produces not the amount of insulin he has as a whole. If we measure insulin it's going to be inaccurate because we give him exogenous insulin as well. But remember we said there's something which is produced in equal amounts with insulin and it has a longer halflife which is a ceptide and so it's the best marker of indogenous insulin.
Okay.
Now let's talk about glucagon. Glucagon is the opposite of insulin. It's the catabolic or the fasting hormone. The source is the alpha cells of the pancreas. A trigger which uh results in the release of glucagon is decrease in blood glucose, increase in amino acids and increase in stress. Now if you can recognize this amino an increase in amino acids is common in both. So when you get increased amino acid or when you eat a protein richch meal, you're going to increase both insulin and glucagon for different reasons of course but you're going to still increase insulin and glucagon.
Um and the effects of insulin is catabolic which is breakdown or releasing and uh we we spoke about unique thing related to insulin receeptide. The unique thing over here with glucagon is okay I'm not going to point down just focus on the last one which is stress.
um in times of stress or when the body is stressed out, you release epinephrine and norepinephrine, the catacolamines.
And what they do is that they override insulin even if the blood glucose is um high. Okay, low, high, whatever, the body doesn't care if you uh release epinephrine. This is going to override the function of insulin and you're going to stimulate glucagon and then glucagon is going to release glucose. Okay. So, usually we release uh epinephrine norepinephrine where in when we're like in sports or when we're stressed out when we need the energy basically and that's why insulin function is overridden by um the epinephrine and we stimulate glucagon and then we increase our blood glucose.
And now this one says regard uh they will both work against their triggers.
Let's look at the triggers of insulin and see what's going to happen. So a trigger of insulin is increase in blood glucose. So insulin is going to try to decrease the blood glucose. Amino acids increasing amino acids is going to trigger insulin. So insulin is going to reduce the amino acids and it does that by increasing protein synthesis and incretins don't worry about that.
Now for glucagon decreasing blood glucose it's going to try to increase the blood glucose by releasing glucose increasing amino acids is going to decrease or try to aim to decrease amino acids um for example one example I can think of is using the amino acids in gluconogenesis for example and stress don't worry about >> [snorts] >> Now, let's quickly review some of the anabolic and catabolic reactions. Um, you don't really need to know the details. I just want you to remember them from ML one, okay? Cuz it's going to help you with the functions of insulin, functions of glucagon, metabolism, and diabetes, um, all of that later. Okay? So, the first one is glyco. By the way, most of the anabolic reactions are going to be promoted by insulin except gluconioenesis and most of the catabolic reactions are going to be promoted by glucagon. Um, is there any exceptions? There is no exceptions.
So, gluconioenesis if gluconogenesis is put over here this is going to be insulin reactions, glucagon reactions. Okay.
So, let's uh talk about each reaction individually. Glycogenesis it means formation of glycogen we are building glycogen it's anabolic then you have protein synthesis again anabolic is you're building and then tagg synthesis which is the triasyl glycerol synthesis you're building the three fatty acids and glycerol backbone thing it's anabolic then gluconneogenesis you're forming new glucose and therefore it's anabolic And then you have the catabolic reactions starting with glycogenolis.
Break it down. Glycogen lis means you're breaking down glycogen. Catabolic ketogenesis.
This is formation of ketone bodies. Now even though it's formation, you form it from fatty acids and fatty acids are larger. So you're going to get the fatty acids break it down and this is going to form ke ketones. So even though it's a formation reaction kind of, it's still catabolic.
It's an exception. Just memorize it.
Then you have fatty acid oxidation.
You're breaking down fatty acids. This is catabolic. Then protein breakdown.
Catabolic. Lipolyis is also catabolic because you're breaking down lipids.
Now um glute transporters very important, very high yield. Don't worry about glute 3 and glute 5 though because the remaining are very important and very very high yield. The first one is glute one and it's found in RBC's and in the bloodb brain barrier. Okay.
The second one is glute 2 and it's a glucose sensor. It's found in the liver and in the pancreas in the beta cells.
So the beta cells are going to have this glute uh two. They can sense the glucose level in the blood. when it increases a lot they can release insulin. Okay. And then glute three skip. Glute four is insulin dependent and it's found in muscles and in adipos tissue. And the importance of this is that when insulin rises it means basically it goes from glut to glute 4.
First of all the sequence starts when we increase our blood glucose. This is detected by the pancreas through the glute 2 sensor and then it it's going to release insulin.
This insulin is going to go through the blood. It then goes to the muscle uh cells or the atipose tissue and then it's going to recruit the glute 4 to the cell membrane. And when it recruits the glute 4 to the cell membrane, this is then going to allow glucose to enter the cell. And therefore, this is insulin dependent. It's not always there. It's only going to be there when there's insulin.
And then we have glute 5. Don't worry about glute 5. So if a question asks you about um which of the following uh tissues are insulin dependent or they absorb glucose uh when insulin is present and they give you liver, pancreas, um RBC's, the brain, uh muscle, what's the answer going to be? The answer is going to be muscle because the others are not insulin dependent. They're insulin independent. The only one which is insulin dependent is the muscle from these options also adapostium.
Now let's compare insulin and glucagon.
If I were to put stars like molecularis I put five stars over here, five stars over here. Very important slide.
[snorts] Okay. So let's start with insulin.
Insulin promotes glucose uptake. We spoke about this. It increases the glute for um expression and glute for transporters move to the cell membrane and they allow glucose to enter the muscles and adipost tissue.
Insulin also increases protein synthesis because it's an anabolic uh process and as we said before insulin and gluccoone both act against their triggers. One of the triggers was increased amino acids in the blood. So when we have increased amino acids, we increase the protein synthesis and then um insulin also increases glycogen synthesis because again it's a anabolic hormone. So anabolic simply think of it this way that insulin and glucagon. Insulin is when you're overfed. Insulin tells your body you have so much let's store. And glucagon does the opposite. It tells your body you're starving let's release some sugar into the blood.
Okay. So, um, where were we? We're over here.
Okay. Insulin increases glycogen synthesis, also known as glycogenesis.
Um, we spoke about it before. Pretty straightforward. And then fat synthesis.
Insulin increases fat synthesis.
And it does this by increasing LPL. You should review this in uh your fat lectures, but uh LPL basically increases when we're building up fat. And then HSL increases when we're breaking down fat.
So LPL increases with insulin.
Uh HSL decreases with insulin. LPL decreases with glucagon. HSL increases with glucagon.
Um, okay. Let's get back to insulin.
Insulin increases fat sensitives because it's anabolic and then insulin decreases ketogenesis.
Um, you just have to memorize this. You don't need to produce ketone bodies. And we're going to talk about this later in um which lecture feed fast. Okay? So, ignore this for now.
And then um insulin decreases lipolyis.
You don't need fats in the blood. You already have so much glucose. you instead want to build um fats. You don't want to break down fats.
And then you have gluconneogenesis which is decreased when insulin is released.
And it's pretty straightforward. You have so much glucose in the blood. You don't want to build new glucose.
And glycogenolyis is also decreased. You already have so much glucose. You don't want to break down glycogen to give you even more glucose.
And now glucagon is basically going to do the opposite of these functions. It's not going to affect glucose uptake because it's not going to affect the glute for transporters.
Uh glucagon is going to decrease protein synthesis because um it wants to release these amino acids into the blood to the liver for the liver to uh use them in gluconogenesis to produce new glucose. But it's not included here.
It means it's not important for you guys.
And then glucagon decreases gluc glycogen synthesis again it decreases glycogen synthesis because or glycogenesis because we don't have that much glucose in the first place we're not storing now we are releasing and glucagon decreases fat synthesis because again we spoke about the LPL HSL remember this insulin is going to increase LPL activity insulin is going to decrease HSL activity glucagon one is going to decrease LPL activity. Glucagon is going to increase HSL activity.
And LPL is used for fat synthesis, lipoprotein lipase. HSL is going to uh is used for lipolyis.
Okay. [snorts] Um okay. So glucagon decreases fat synthesis and glucagon increases ketogenesis. And we will talk more about this in the feed fast cycle.
Glucagon also increases lipolyis and glucagon increases gluconogenesis.
The reason it increases gluconioenesis is because again glucagon senses that we're starving. The the cells need glucose and so it's going to tell the liver we need glucose no matter what produce new glucose. Therefore it's going to increase gluconneogenesis formation of new glucose. And it's also going to increase glycogenolyis which is producing glucose or breaking down glycogen to give us glucose because again we need the glucose. And finally it's going to increase amino acid uptake. Just memorize this. Okay.
Now what about protein breakdown? It's not included in this. Would insulin increase protein breakdown or not?
The answer is no. Insulin increases protein synthesis. Glucagon would increase protein breakdown. And I kind of gave you the reason before. It's because we want the amino acid precursors to be used in gluconogenesis.
So we're going to break down proteins, get the amino acids, put them in the liver, and then produce new glucose.
Okay. Now let's move on to question one.
Which of the following best explains why ceptide levels are measured clinically to assess endogenous insulin secretion 3 2 1 There's not much explanation to do here. The answer is C. Uh C pepide is secreted in equimar amounts with insulin but has a longer halflife. That's why it's the best uh marker for indogenous insulin.
Question two. During periods of physiologic stress, such as fight or flight response, which of the following best describes the interplay between epinephrine and glucagon?
[snorts] 3 2 1.
Okay. Is it a epinephrine suppress glucagon? No. Does the opposite actually. Epinephrine enhances insulin?
No. Does the opposite. Glucagon inhibits epinephrine release? No. Glucagon and epinephrine both promote insulin mediated glucose uptake. No. Um, epinephrine stimulate glucagon levels overriding the usual feedback from blood glucose levels. Correct. The answer is E. So the answer is C over here. The answer is E over here. Question three.
Which of the following is a correct effect of increased insulin levels?
Increased gluconogenesis in the liver.
decrease glycogen synthesis in muscle, decrease hormone sensitive lipase activity in the adiple tissue, increased lipolyis in adiple tissue or decrease glucose uptake by cells.
One of them is the effect of increased insulin. The others are the opposite. So let's go one by one. Increased gluconogenesis in the liver. This is the opposite. Uh the ins insulin decreases gluconogenesis.
decreased glycogen synthesis in the muscle. Incorrect. We do the opposite actually. We want to store the glucose because we have so much of it in the blood. So, we're going to store it in forms of gag.
And then C um decreased HSL activity in adipos tissue. We said HSL breaks down is involved in lipolyis. So, this is correct because we don't want to break down fats. we already have enough fuel in the body.
And then D is increased lipolyis in adipos tissue. Incorrect. It's decreased lipolyis. And then E is decreased glucose uptake by the cells. It's incorrect. We said the first point was increased glucose uptake.
Now lecture 11 feed fast cycle.
[snorts] I say we just go straight into it. Okay, let's start. So for the feed fat cycle we have three main phases which you should know in details. The first one is the absorptive or the fed state and you should know both names by the way for each state. [snorts] The absorptive or the fed state and it's uh the first 3 hours after you eat the meal. So the hour zero is going to be the moment you finished your meal and then up to 3 hours after the meal. And in this state, you have a high insulin, low glucagon ratio. So an increased insulin to glucagon ratio. And you're in the storage mode. You're basically storing uh glucose, uh fats, whatever. [snorts] And then in stage two, the postabsorptive or the early fast stage, um it's going to be 3 to 12 hours or maybe even up to 18 hours after your meal. And in this stage insulin drops, glucagon rises. So the ratio of insulin to glucagon is going to decrease a little. And the main aim of this is to maintain the blood glucose and to mobilize some glucose to release some glucose into the blood. Okay. [snorts] And then uh the last stage is the prolonged fasting or starvation and it's more than 24 hours. And in this stage you don't have any glycogen reserves.
you finished your glycogen, um, glucagon dominates. Insulin is going to be very, very low. Glucagon is going to be very, very high. And some key factors in this is fatty acids and ketones. They play a huge role in this stage. And you're just trying to adapt to whatever fuels you have remaining.
So, let's talk about phase one, the feds phase or the fed state. And as we said, it's the storage mode. So let's focus on what happens in each of those organs.
Uh the liver increases glycogenesis.
That's the first thing. It also increases fatty acid synthesis using the excess glucose. So we're just storing the glucose basically.
um and it increases and wait fatty acid synthesis using the excess glucose and then it releases those fatty acids into the blood through the kyomicrons and VLDLS for the adipos tissue to do the remaining. Okay. And then let's talk about the muscle. Muscle uh muscles increase the glucose uptake. We spoke about this before. Muscles increase the glucose uptake glute 4.
um they increase glycogenesis and protein synthesis. So they're storing glycogen and they're uh forming proteins now. And one thing about muscles which you should know very important is that they can store glycogen but they cannot release glucose and this is because they lack glucose 6 phosphatase which sets glucose free. So even if they try to release the glucose into the blood when um we're in when we're starving, they cannot do that. Okay. And then we have adipose tissue and adipose uh tissue increases glucose uptake again glute for transporter and TAG storage.
So they form triglycerides and they store them using the glucose the excess glucose we have in the blood for glycerol and the fatty acids from the kyomicrons and the VLDLs.
That is it for adipose.
And now for very very very key uh players in this entire feed fast cycle the brain and the RBCs. Um, now the brain and the RBC's use glucose normally because this is what they can use. They can't use fatty acids both of them. But we're going to see something interesting about the brain in the last phase.
And if you remember, we said they are insulin independent. And what's their transporter? Glute one. Glute one for the RBCs and for the bloodb brain barrier.
Now question what's happening to LPL activity and HSL activity in phase one 3 2 1 Okay remember we said phase one is insulin dominated insulin is high glucagon is low so the effects of insulin are going to take place and we said insulin decreases HSL activity and it increases LPL activity because we don't want to break down fats instead we want to store them and therefore this is the answer.
Now let's move on to phase two the early fast and this is when we start to um the glucose in the blood is is basically running out and we're releasing glucose now. So in the first stage of phase 2 like the first part of phase 2 we said it's 3 to 12 hours maybe even up to 18.
Let's say we're like in the 3 to 6 hours. In that first stage, you're going to have glycogenolyis because we still have glycogen reserves.
And then after the glycogen reserves run out, we rely on gluconogenesis.
Again, one more time. In the beginning, we use glycogen reserves, release uh break down the glycogen into glucose and release it into the blood. And then we rely on gluconneioenesis because we don't have any more glycogen. we have to form new glucose using fatty acids.
Okay.
Uh uh I mean sorry uh using the amino acid precursors we spoke about before.
And in this stage the liver survives using fatty acids. So the liver is since it can use fatty acids unlike the brain and the RBCs it uses those fatty acids and it releases this glucose into the blood. Okay it's basically being generous. [snorts] And then for stage two, the muscle um muscles increase fatty acid oxidation.
This is the first part. They increase fatty acid oxidation. So they're not using glucose anymore. They're they use uh fatty acids and they release amino acids to support gluconneogenesis.
So now as you can see all of the organs are thinking of the the the body the body tissues which cannot use anything else but glucose and they're they're shifting their fuels from glucose to fatty acids and they leave the glucose to the others. But remember we said the muscle can store glycogen. Let's go back. We said the muscle can store glycogen but it cannot release it. So in the first stage of in in phase two they can also use this glycogen reserves but again they cannot really release it. So they use this glycogen reserves and they use fatty acids but they also release some amino acids to support gluconogenesis.
Now um for adipos tissue they switch from storing to releasing now. So they increase lipolyis that's the first key part and then they release those fatty acids for energy and the glycerol for gluconogenesis.
So they release those fatty acids for the body parts or the tissues which can use fat for energy and they releases glycerol for the liver uh to using gluconogenesis and uh the this overall shift uh helps keep glucose for brain and the RBCs. Now let's come down to the brain and the RBC's again they use glucose normally because they don't have any other choice. Uh they're also insulin dependent. So the rate of using glucose does not change throughout phase one or phase 2. Okay. [snorts] [clears throat] Now what's happening to the LPL activity and HSL activity remember we said in the beginning insulin was dominating and therefore insulin activity in phase one I mean therefore HSL is going to decrease LPL is going to increase. Now it's the opposite because as you can see we are um for example focus on the adapost tissue it's increasing lipolyis it's breaking down fats so clearly we're shifting from the insulin to the glucagon and so again what's happening to LPL activity and HSL activity LPL activity is actually going to decrease and HSL activity is going to increase because again we're mobilizing ing those fatty acids. We're not storing them anymore.
And now for the last phase, phase three, starvation. Uh the body is just trying to adapt to whatever it has. And this is where most of the questions are going to come from. Very high yield, very complicated, uh I should say. So please focus.
In phase three, the liver purely relies on gluconogenesis because glycogen is fully depleted.
That's the first thing. Second thing is it starts ketogenesis. It produces ketone bodies and it does that as we said before using fatty acids. So it's going to use fatty acids turn them into ketone bodies.
But again even though the liver can produce ketone bodies it cannot use them because it lacks the enzyme thofurase.
That's a question. Okay remember it.
It's probably a question.
and the liver uses fatty acids instead in this stage. It also uses a little glucose but mainly fatty acids. We have a summary table for all of the you uh the organs and what they use. So you can memorize it from later. And then muscles increase fatty acid oxidation even more.
We said they use fatty acids but they increase fatty oxidation even more [clears throat] and um they release more amino acids to support gluconogenesis. So, there's muscle wasting in this stage. You're going to basically go from a buff guy to a skinny guy because you're just trying to survive.
Um, now even though the muscles can use some ketones, they prefer to leave most of the ketones for the brain. We're going to talk about this in a bit. And finally, or before we get into our BCS and brain, we have adipose. And adipos tissue increases lipolyis even more. So whatever we saw in phase two, it's being exaggerbated times two. Think of it like that. So adiposition increases lipolyis even more and they release those fatty acids for energy and the glycerol goes for gluconogenesis at the liver. And again we said most tissues can use fat for energy. It helps keep the glucose for the brain and the RBCs.
And finally, for the RBCs in the brain, RBC's rely 100% on glucose because they lack mitochondria. It's not like they have a choice. They can't really function on fatty acids or ketones or whatever. They can only use glucose. No glucose, they're going to die. And so, the entire body, as we can see, is trying to leave this glucose for the RBCs and the brain. And now, let's talk about the brain. Surprisingly, the brain now uses ketones and glucose.
And so this makes sense on why the liver starts ketogenesis because the brain can actually use ketones.
So the brain shifts its focus from glucose only to ketones and glucose. And this is to leave some of the glucose for the RBCs because the RBC's cannot function on any ketones any uh thing other than glucose.
And another reason you may wonder why why the brain can use ketones and not fatty acids. It's because fatty acids cannot cross the blood the bloodb brain barrier. Okay?
So once we change those fatty acids to ketones, now the brain can use it. Now keep in mind even though the brain uses ketones, it still uses some glucose. So if a question uh asks you um does the brain use glucose in phase three, your answer should be yes. What is the major uh energy source for the brain in phase three? It's going to be ketones.
Do you get the difference? Okay. Now, what's happening to LPL activity and HSL activity? We said that phase 2 and phase three phase three are kind of similar because you're just mobilizing and releasing um glucose and whatever energy source you have. So, the answer is the same. This is going to be to a larger degree. So, LPL activity decreases even more. HSL activity increases even more.
Now this is a very very important summary table on what every organ uses.
I'd like you to pause read it on your own. Nothing new is mentioned over here.
Just pause and read it on your own.
Let's move on to question one. Which of the following will be the source of energy for the brain during starvation?
So the answer over here is D. Now the question clearly is trying to get from us what is the main source of uh energy during starvation.
So even though we have glucose as one of the options, the main source is ketone bodies. And uh they might try to question you um or trick you. You guys should know this by now. The acettoacetate is um used in keto it's one of the products of ketogenesis all of that. So they might question you like what increases in the blood in person during starvation. Acettoacetate um acetone ketone bodies ketogenesis all of those are keywords. Okay they both they all mean the same thing.
Um question two now. Which one of the following tissues relies only on glucose for ATP production?
321. The answer is E. Ariththraittytes is another name for RBCS.
Question three.
During fasting, muscle glycogen cannot provide glucose to the blood. Which of the following is the most probable probable explanation?
3 2 1 the answer is C. Muscles lack glucose six phosphotase and you just have to memorize it honestly.
Okay. Now for the last lecture bear with me guys. But this lecture is very very simple. I think I have like four slides for it only.
Now starting with the easiest stuff blood glucose. The normal levels of fasting plasma glucose is 70 to 100 milligrams, excuse me, is 70 to 100 milligrams per deciliter. Okay?
Any less than that, you're in the hypoglycemia playground. Any more than that, it's hyperglycemia.
Now, if you have more than 126 milligs per deciliter on two separate occasions, you can you're diagnosed with diabetes.
Okay?
type one, type two, that's not our concern. You're diagnosed with diabetes.
And so from this, we can learn that if diabetes is not um treated, you're going to have hypoglycemia.
Now, let's look at the HBA1C, our best friend, um and when you have HBA1C level of 6.5% or higher, you're a diabetic.
Okay? Anything slightly lower than that is pre-diabetic. and then even less you're normal okay or hypoglycemic depends don't worry about the values except the ones I've shown in this uh slide now the best indication um of increased blood glucose over weeks is hbaw1c because it gives an average for the last 3 month it doesn't give normal fasting plasma glucose gives an average for the um it doesn't give an average it gives your current status but hbaw1c basically snitches on you. It tells the doctor what's been happening the past 3 months because this is the RBC lifespan and HBA1C measures glycated hemoglobin.
Okay.
Um and lastly, high blood glucose causes the glycation of many proteins and this causes retinopathies which is eye problems, nephropathy which is problems with the nephrons, kidneys basically and neuropathy neurons neuron problems.
[snorts] Now this is a very very very this is basically the entire lecture in one table. If you can pause and read it and with this as well, you're done for this lecture almost except for one clinical case. So let's compare type 1 and type two diabetes. Type 1 diabetes, the patients are usually young and thin.
Type two diabetes, the patients are usually old and obese. [snorts] Type two diabetes uh is characterized by absolute insulin deficiency. Type two diabetes you have insulin resistance and in the later stages you have insulin deficiency.
Type 1 diabetes is um basically autoimmune attack on the beta cells and it's triggered by a virus and even though there is a genetic influence it's weaker compared to type 2 diabetes.
And then uh the path pathogenesis for type two diabetes is not completely understood but they say that it's due to the increased adipose tissue and therefore there's more inflammation which causes insulin resistance and then since the cells the muscles the adipose um are insulin resistant the beta cells must produce more insulin and this is going to work for some time. They're going to produce more insulin, more insulin, more insulin, and then they're going to basically uh die or become or lose their function. Like they can't keep producing infinite insulin. So, at some point, they're not going to work anymore. And this is where you start experiencing the symptoms. This is late stage type 2 diabetes and this is where insulin deficiency happens. Okay.
Um and then you have insulin therapy required for type one always like type one you have to give them insulin. Type two is different. Insulin is only required in the late stage because as we said in the beginning even though there is some insulin resistance it's being compensated by the overp production of the beta cells. Once the beta cells begin dying you have to give insulin.
Um and then presentation of type one is polyura, polyypipssia and polyphasia.
Let's try to understand them. Polyura is frequent urination. And the reason this happens is because as we said with both types of diabetes, you have hypoglycemia.
In hypoglycemia, you have increase in blood glucose. And if you go back to renal, you can remember that glucose is like always always being removed from the urine added to the blood. And we never go over that value. Just think of it. If you have so much glucose to the point that the um I don't want to give you complicated names, but if you have so much glucose to the point where we cannot recover all of it, then the glucose is going to start appearing in the urine. And then one thing you should know about glucose is that it pulls water with it.
So quick recap, glucose is increased so much, it spills into the urine, it pulls some water with it and then you have frequent urination.
This links us to the second symptom which is polyypsia which is uh thirst basically you have to drink you're always thirsty you always want to drink water and this makes sense because you're losing so much water in your urine. So polyuria, polyypipssia, they're both linked together. And then polyphasia is because you don't have insulin. Um you don't your your body is not building muscles, your body is not building fat. You're always in this stage of glucagon and releasing and uh you need energy. So you're going to be hungry.
Um and as for type 2 diabetes, it's usually detected by routine screening.
However, in extreme cases when the beta cells die, you can present with some of those. Okay.
Now, um some key factors for type one compared to type two is that there is no hyperinsulinemia. You don't have increased insulin. Actually, you don't have any insulin.
Um and you also have keto acidosis because uh as we said there's no insulin and therefore you have glucagon.
Glucagon as we mentioned before in phase three um results in ketogenesis and this increased ketones in the blood is going to have ke is going to make the patients present with ketoacidosis. Okay. And lastly we have hyper lipidmia in type one. Hyper lipidmia is not present in type two. So quick recap on this last one because you can answer so many questions if you just memorize this. No hyper lipidmia ketoacidosis uh sorry no hyperinsulinemia keto acidosis and hyper lipidmia are found in type one. Type two there is no keto acidosis. You have hyperinsulinemia and there's no hyper lipidmia.
that is 90% of uh metabolism and diabetes. Now there's one thing which I really want to talk to you about especially since we're fasting now uh which is diabetic hypoglycemic attack.
Usually when you're fasting and then you eat a meal the insulin overshoots. Okay.
And this is normal. It's known as postrandrial or reactive hypoglycemia.
insulin overshoots and then the glucagon is going to fix it back. Okay, so the levels go back to normal. This is in a normal individual, but in a type 1 diabetic specifically, this doesn't happen. So the patient would usually present with um them taking their insulin shots or insulin uh IV injections, but they forget to eat. So think about it. They get insulin exogenously, of course, they forget to eat. the body now thinks that oh we have insulin it means we have so much glucose let's absorb all of the glucose and then we're the patients would present with severe hypoglycemia now normally glucagon would compensate but in individuals with long-standing type 1 diabetes the alpha cells which produce glucagon are also dead so you don't have glucagon you don't have insulin with so much insulin just not being opposed you're going to have severe hyper glycemia and it's a medical emergency. Okay.
Now let's talk about the treatment of type one and type two. Type one has no treatment. The standard care is giving insulin. Okay. Just memorize this. Type two for newly diagnosed diagnosed patients. You can save them without you can save them by decreasing body weight because decreasing body weight has been shown to improve glucose tolerance and insulin toler uh insulin uh sensitivity was the word resistance. So it's going to reduce the insulin resistance and increase uh improve glucose tolerance and exercise and dietary modifications also are important for newly diagnosed patients. As for late diagnosed patients, it's it's it's not it's not going to work as much. It's still beneficial, but you still need medications.
Now, some anti-diabet diabetic agents for type two. Remember we said type one, just give them insulin. Type two, you can give them two drugs which are metformin and sulfonyluras.
Metformin works by decreasing the glucose production. overall this is the overall function and it does that by inhibiting gluconioenesis in the liver.
Okay. So if you were to memorize one line for metformin it's going to be this one inhibit gluconioenesis and therefore decrease glucose production.
Um and the other one is sulfonyl uras and they work in a different way. They basically their over like the end end stage or the end result they reach is increasing the insulin secretion and they do that by blocking the ATP sensitive potassium channel also known as KTP and then this results in uh deolarization of the beta cells. when they're depolarized. You guys should know this by now. At the end of the nerve, the last stage of deolarization, calcium moves in and then calcium moves in causes the vesicles to fuse with the plasma membrane, release the insulin.
Okay, so quick recap. Sulphonalures uh sulfonylurases increase insulin secretion and they do that by blocking KATP which depolarizes the beta cells and releases insulin into the blood.
And lastly, let's talk about gestational diabetes. Again, I told you guys before that the vibe I'm getting from your exam is clinical oriented. So, try to focus on your clinical aspects of your lectures. Um, gestational diabetes. So, this is when the mother has uh diabetes when she's basically pregnant. And it doesn't have to be like type one, type two.
uh some uh in some cases they they develop diabetes while they're pregnant.
Basically increase blood glucose because they're trying to feed their babies and then in some cases even after the delivery it remains and the mothers become diabetic. That's not a concern over here. Just remember gestational diabetes has severe consequences and uh a consequence is fetal macrosia which is a large baby as we can see over here.
mashallah. And they're also shaky. The reason they're large is due to the anabolic effects of insulin. As we said, um insulin causes the uh storage and the production I mean storage and the building of stuff. So the baby over here is clearly large.
This is due to the anabolic effects of insulin. And the baby is also going to be shaky. They're going to be hypoglycemic. They're going to shake.
Okay.
And this is purely due to the honestly you just have to memorize this. It's due to the baby's relative hyperinsulinemia.
So since the mother has a very high blood glucose level, the baby is also going to produce a lot of insulin to counter that. So once the baby has been delivered, the baby is going to experience hypoglycemia and this is due to the baby's relative hyperinsulinemia.
And question time. Other than obesity and genetic profile, what is the most important thing in telling that it is type 2 diabetes?
3 2 1. Remember we said that type two diabetes is classified by insulin resistance and in later stages insulin deficiency.
So other than obesity and genetic profile, the answer is going to be muscle resistance to insulin. The answer is E.
Question two. What is the function of metformin in a diabetic patient?
The answer is a decreases gluconogenesis by the liver.
You just have to memorize it honestly.
Nothing much.
You know what? Okay, let me give you a question on this. Uh what is the function of sulfonile urases in a diabetic patient?
If this was an SEQ, what would you write?
Your answer should be it inhibits the potassium ATP channels. Um and then this is going to depolarize the beta cells and this is going to result in calcium entry, vesicle fusion with the plasma membrane, release of insulin and that is it. Increased insulin production overall.
Question three. A 12-year-old boy presented with fatigue, polyypipssia, polyura, and polyphasia. A finger sick glucose measurement shows a glucose level of 350 mgs per deciliter in his serum. He's diagnosed with type 1 diabetes. Which of the following which one of the following is most likely occurring in this patient?
It's pretty complicated. I'll give you 5 seconds. You can pause, of course.
5 4 3 2 1. Okay, let's go through it.
Type 1 diabetes. So will there be a decrease in hippatic gluconogenesis?
Not necessarily.
Um you don't have any insulin. So it's going to be the opposite. You're going to have increase in gluconogenesis.
Um then B is insulin resistance. No, we said type one. There's no insulin resistance. You don't have any insulin to begin with. And then C is decrease conversion of fatty acids to ketone bodies. Not really. there's an increased conversion of fatty acids to ketone bodies and that's why we have keto acidosis in type 1 diabetes patients and then D increased production of acettoacetate and remember this is how they like to trick you it is correct um acettoacetate ketone bodies ketones acetone keto acidosis they're all the same okay and then lastly increase stores of triacyl glycerol and adipos tissue.
You're not storing, you're releasing, you don't have any insulin. Okay.
Um, this is the answer key. Uh, try to solve the questions without looking at the answers. This is the only reason I have them at the end. I know it's not very convenient, but it's a review. You guys should aim to answer all of the questions. And if you've watched till the end, thank you very much. That is all. I wish you guys the best of luck and just try to push through this burnout season. Don't underestimate maul, but don't sleep on msk as well. If you have any questions, since I had to basically study for your midterm again, feel free to ask me. Even if it's uh a low yield point, feel free to ask me.
And thank you very much.
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