Misc

• Ways we move nitrogen around

  • transamination pairs

  • nitrogen by itself is really toxic

  • predominant form is $N{H}_4^{+}$$NH_4^+$ , not $N{H}_3$$NH_3$

    • $p{K}_a$$pK_a$ of $N{H}_4^{+}=3.9$$NH_4^+ = 3.9$ , so at physiological pH , its protonated

  • transamination in most tissue is taking the amino acid through a transamination and producing an alpha-keto-acid

    • also at the same time taking the alpha-keto-glutarate and making glutamate

    • glutamate is then converted into glutamine

      • we do this via glutamate synthatase

        • this incorporates an additional $N{H}_4^{+}$$NH_4^+$

      • one of the few enzymes that allows nitrogen fixation in humans

      • ATP is used for energy

  • most extra-hepatic tissue , except for muscle does transamination

  • we transport the glutamine in the blood with 2 nitrogen’s

• In muscle tissue ,

  • as the muscle cells are “chugging through” amino acids , we do a similar reaction to transamination

    • except pyruvate gets converted into alanine

      • alanine then goes to the liver

        • it then undergoes transamination to make pyruvate

          • we have now moved the nitrogen to the liver

          • most likely creating glutamate

        • the pyruvate most likely goes to gluconeogenesis , because if you are breaking down muscle , you are in the fasting state

        • that glucose can go back to the muscle cells

          • most likely , it goes to the brain though

        • this is called the glucose-alanine cycle

        • the carbon is really coming from stored glycogen in the muscle cell

• Glycogen Storage Disease :

  • they can’t convert glycogen to pyruvate

    • starving off the carbon for the glucose-alanine cycle

    • decreases their ability to undergo gluconeogenesis because one of their precursors is disrupted

    • Lactate

    • Red blood cells produce a ton of lactate

• Glucose-alanine cycle is not “closed”

Enzymatic Transamination

• Requires PLP for amino transferase reactions

The Glucose-Alanine Cycle

• Most of the time its glutamate that is made

• not a “closed cycle”

Misc

• We are pumping in alanine from muscle tissue

• $N{H}_4^{+}$$NH_4^+$ is released from glutamine and glutamate

• Glutamase = gets of first nitrogen

• Glutamate dehydrogenase = gets off second nitrogen

• Nitrogen in the blood is toxic ,

  • alanine and glutamine concentrations are really high because they are transporting nitrogens to the liver

• First enzyme in the cycle , carbamboyl phoshpatase-1 is part of urea cycle

  • the “2” is part of amino acid degradation

• enzymes are located in the mitochondria

• citruilline is transported out of mitochondria

The Reactions in the Urea Cycle

• $N{H}_4^{+}$$NH_4^+$ = first source

• Aspartate is the second source

• arginine is a pseudo essential amino acid

• anytime you have high nitrogen turn over , arginine gets stuck in the cycle

• Hans Krebb discovered the TCA and urea cycle

• google the “krebbs bi-cycle”

• huge link between these 2 cycles

• once we have arginine , we release urea through arginase

  • reforms arginine to complete the cycle

• Remember :

  • what are the 2 sources of nitrogen

    • $N{H}_4^{+}$$NH_4^+$ and aspartate

  • what is the enzyme that forms urea ?

    • arginase

      • gets rid of 2 nitrogens

  • how is it regulated ?

    • don’t want to turn off any enzymes

    • in the urea cycle = feed forward cycle

      • if the metabolites are there , it goes

        • we have to get rid of nitrogen

      • urea is really soluble , but still kind of toxic

      • we don’t want anything to back up

    • in times of high nitrogen turnover , how do we upregulate the urea cycle ?

      • allosterically regulate the first reaction

      • as glutamate is being produced , it encourages N-acetyglutamate synthase

        • allosterically upregulates carbamoyl phosphate synthetase I

• there are people deficient in urea cycle enzymes

  • CPS1

  • if you are deficient , you die very young

    • that nitrogen will build up as $N{H}_4^{+}$$NH_4^+$ in the liver

    • the liver will will secrete it ( $N{H}_4^{+}$$NH_4^+$ ) , it gets into the blood

      • causes mental defects

    • Treatment :

      • we give them small organic compounds that the liver takes up and react with the ammonia to bind and helps get rid of it

      • extremely low protein diet

        • 1 hot dog is too much

      • even a common cold cause spike nitrogen turnover , and send them to the hospital

Aspartate–Arginosuccinate Shunt

• there are common intermediates between Urea and TCA cycle

• ornithine and citrulline need to be transported across mitochondria

  • moving around a ton of carbon back and forth

Questions

1. The electron transport chain directly produces ATP ?

   • False , it uses indirect substrate level phosphorylation

2. The urea cycle is considered a feed forward cycle ?

   • True

3. Which of the following amino acids can donated a carbon to folate?

   • Serine

4. Which of the following amino acids is catabolized to acetyl-CoA?

   • Leucine

5. What are the two direct sources of nitrogen for the urea cycle?

   • $N{H}_3$$NH_3$ and aspartate

6. What two molecules primarily transport nitrogen to the liver for the urea cycle?

   • Glutamine and Alanine

Glucose

• Glucose —> 2 Pyruvate : substrate level phosphorylation

  • Yields a net of 2 ATP , 2 NADH

• 2 Pyruvate —> 2 Acetyl-CoA

  • Yields 2 NADH

• Acetyl-CoA —>

  • Yields 2 ATP

  • 2 FADH2

  • 6 NADH

• 10 NADH = 25 ATP

• 2 FADH2 = 3 ATP

• 2 ATP = 2 ATp

• = 32 ATP

• TCA Cycle = phos

• C16 —>

  • 8 Acetyl-CoA

  • 7 FADH2

  • 7 NADH

• 8 Acetyl-CoA —>

  • 8 ATP

  • 24 NADH

  • 8 FADH2

• 31 NADH = 77.5

  • investment phase happens pre oxidation , so minus 2

• 108 ATP - 2 = 106 ATP

1. These pathways energetically mean nothing unless we continue it on through the electron transport chain

2. 31 NADH = 77.5 ATP

   • what is this telling us ?

     • its an indirect process , its not a substrate level phosphorylation

     • the energy we get back from NADH and FADH2 is not a direct process

   • Complex 5 = makes ATP

   • electron transport chain is raising the water behind the dam

   • oxidative phosphorylation

   • because they are indirect processes , there are caveats

     • something can disrupt it

   • with substrate level phosphorylation , you either make ATP or you don’t , nothing can really disrupt it

3. The numbers are averages ( 2.5 , 1.5 )

• for glycolysis its 30 or 32

  • we have to deal with the NADH

  • the cytosolic NADH can not cross into the mitochondria

    • it uses 2 different shuttles

      • the malate-aspartate shuttle

        • inner membrane space ➡️ the matrix

        • outer membrane is really permeable

        • inner membrane is not really permeable

        • in the inner membrane space , you take aspartate and make oxaloacetate

        • the oxaloacetate can then go make malate

        • in this process we take NADH and move electrons onto malate , regenerating NAD+

        • malate is transported across the membrane

        • you then reform oxaloacetate , taking an NAD+ and make NADH

        • you reform aspartate and transport it across the membrane

        • oxaloacetate can’t be transported across the membrane

        • moved an NADH across , but nothing really happens , you reformed it

          • energy doesn’t change

  • the other way it gets across is the glycerol-3-phosphate shuttle

    • we go from DHAP to glycerol-3-phosphate

      • in this case , we take the NADH and make NAD+ again

      • the glycerol-3-phosphate can go back to make DHAP

        • there’s an enzyme glycerol-3-phosphate-dehydrogenase on the mitochondrial membrane ( membrane-bound )

          • it uses FAD to form FADH2 to produce QH2

      • we lost energy because we went from NADH to FADH2

        • their potential energy is pretty equal

        • however , NADH enters through complex 1 , while FADH2 enters through complex 2. Complex 2 looses more to heat , so therefore less energy

        • if we do this twice , this is how we are loosing 2 ATP

      • if both NADH go through malate-aspartate shuttle we generate 32

      • if both NADH go through the glycerol-3-phosphate shuttle , we generate 30 ATP

• these shuttles bring up something important

  • things going on between cytosolic and mitochondrial matrix

Chemiosmotic Theory

• we take reduced electron donors to pump protons

• pumping protons back to inner membrane space

• figure out experimentally how you can prove that protons are moving ?

  • originally we didn’t know the numbers of ATP generation

    • we originally thought the TCA cycle generated ATP

  • how do we know electron transport chain is just pumping protons , and ATP synthase is generating ATP

Chemiosmotic Theory

• these reaction in the individual complexes are not a one-off reaction

  • not substrate = product

  • they happen at a large scale

  • electrons are getting handed off

  • each complex doesn’t just have one hand off

  • there’s a lot of reactions in glycolysis pathway that don’t do much

  • the same thing is happening here , its a bunch of small reactions

  • all of the complexes contain small molecules in them that are capable of being oxidized or reduced

    • example = cytochromes

      • small proteins that have different hemes in them that can be oxidized or reduced

      • multiple different subtypes of cytochromes

Structure of a Mitochondrion

• snakes back and forth to increase surface area

• tons and tons of enzymes bound to it

• we need a ton of enzymes / proteins bound

• allows more ATP to be produced

Cytochromes

• by changing the ring structure , we change the reduction potential

  • so we have like 100 different reduction potentials

  • we are moving across that reduction potentials

Iron-Sulfur Clusters

• these are the other way’s we move electrons around

• anchored to the protein via cysteine residues

• how they are anchored to the protein , the environment around it , and the complexes on top change the reduction potential

Coenzyme Q or Ubiquinone

• cytochrome-c = mobile electron carrier

  • more hydrophilic

• coenzyme Q = mobile electron carrier

  • more hydrophobic

    • anchored to the membrane

    • has a large hydrocarbon tail , makes it hydrophobic

• acetyl-CoA = “hub”

• complex 1 and complex 2 all dump into co-enzyme Q

• coenzyme q = collection point , and then goes off to complex 3

NADH:Ubiquinone Oxidoreductase (Complex I)

• FMN , iron sulfur clusters

• on the arm , you are oxidizing the NADH to NAD+

• then transferring the electrons to coenzyme Q

  • while doing this you pump 4 protons

  • the Q then goes to complex 3

• electrons from NAHD : complex 1 to 3 to 4

• FADH2 = 2 , 3 , 4

• complexes are not linear , EVER !

Table 19-3

• prosthetic groups have different reduction potentials , allowing for electrons to flow

• the greater the reduction potential , the more thermodynamically favorable it is to flow electrons

• these are not small , very high kilo-dalton numbers

Succinate Dehydrogenase (Complex II)

• also in the TCA cycle

• transferring electron from FADH to Q

• but no electrons are pumped

• produced more QH2 , but haven’t pumped protons

• only oxidizes the FADH2 from the TCA cycle

• the FADH2 from beta oxidation doesn’t happen

  • its only succinct to fumarate

  • the FADH2 from beta oxidation never really produces FADH2 , it immediately makes QH2

• any time you you see FADH2 , its going to be a membrane bound enzyme that dumps it onto QH2

  • means there are others enzymes we haven’t talked about

• Complex 1 , 2 and anything FADH2 linked , they are making QH2. That QH2 then goes off to complex 3

Ubiquinone:Cytochrome c Oxidoreductase (Complex III)

• we are taking QH2 and producing cytochrome c that then goes off to complex 4

• we are pumping protons here

• requires multiple electron movement steps

The Q Cycle

• caveat to complex 3

• this is a theory still

• QH2 is a 2 electron carrier

• Cytochrome C is a 1 electron carrier

• so we produce 2 cytochrome c for each FADH2

  • we don’t know how this works

• through a multi-stage process we produce 2 cytochrome c that are reduced , and then we pump 4 protons

• if starting at NADH , we pumped 4 for complex 1 , for 4 complex 3 = 8

• FADH2 , zero protons from complex 1 , 4 from complex 3

Cytochrome Oxidase (Complex IV)

• we have multiple cytochrome c coming in , but we only pump 2 protons

• take oxygen and produce water

• if we stop oxidizing cytochrome c , we stop oxidizing everything up the chain

• NADH Protons Pumped :

  • Complex 1 = 4

  • Complex 3 = 4

  • Complex 4 = 2

  • Total = 10

• FADH2 :

  • Complex 2 = 0

  • Complex 3 = 4

  • Complex 4 = 2

  • Total = 6

• 6 / 10 = 0.6 = 60%

• 1.5 / 2.5 = 0.6

• NADH = 2.5

• FADH2 = 1.5

  • this is why FADH2 produces 60 percent as NADH does

    • it only pumps 60% of the protons that NADH does

Electron Flow in the Respiratory Chain

• complex 1 and complex 2 produce QH2

Summary of Electron Transport

• at the end of the day 10 protons if you start from complex 1

• 6 if you start from Complex 2

• FADH2 = not a mobile electron carrier

• NADH = a mobile electron carrier

Reactive Oxygen Species

• over nutrition / metabolic syndrome

• we pull metabolism towards ATP

• if we don’t need it , we stop metabolism

• overnutrition is pounding it towards ATP when we don’t need to

• ROS can damage proteins

• we can make hydrogen peroxide

• glutathione can turn it into water

• if you keep “pounding” it , you run out of glutathione or the ability to take care of ROS

ETC

• Complex 5 takes protons back across

• How do we know its ATP synthase and not any of the other complexes that make ATP ?

  • if you disrupt the membrane , there is nothing holding protons back

  • you can make an artificial gradient , add acid to inner membrane space

    • and you will make ATP

  • inner membrane space is more acidic

  • un-couplers will dispel the proton gradient

    • you can have an organic compound that its pKa is around the pH differences

    • on the inner membrane space , it will pick up the proton

      • it then freely diffuses through the membrane

        • and then gives off the proton

      • this is getting the proton through the membrane but circumventing ATP synthase

      • by doing this , you are removing the pH difference

        • ETC still runs , but no ATP is produced

How do we regulate ETC ?

• we pull metabolism by ATP need

• if ATP is high , ADP is low

  • shuts down the ATP synthase enzyme

  • It stops moving the proton across the gradient

  • the rest of the complexes keep building up the gradient

  • high concentrations of ATP cause high amount of protons in the inner membrane space

    • that causes the delta-G of pumping to become positive ( unfavorable )

      • then we can’t pump protons across in the other complexes

        • this shuts down the ETC

          • increase NADH to NAD+ ratio

            • this also turns off the ETC

              • 3 dehydrogenases are sensitive to NADH / NAD+ ratio

                • shuts of pyruvate dehydrogenase ?

                • shuts off beta-oxidation , you don’t have the cofactor

                • glycolysis is iffy , depends on the tissue , but also shuts down probably

• we turn ATP synthase back on when we need ATP again

ETC and ATP Synthase

• if oxygen is being consumed we are running ETC

• if we add ADP and inorganic phosphate , we don’t really do anything

• if we add in succinate , we get a ton of ATP and start consuming oxygen

• Cyanide is an inhibitor of complex 4

  • shuts everything else down

• Part B :

  • proves you need ADP , Pi , and succinate

  • oligomycin is ATP synthase inhibitor

    • stops ETC

  • now add DNP ( an un-coupler ) , dispels the proton gradient

    • red line didn’t change

      • ATP produced

    • but the black line did

      • black line = oxygen consumption

        • tells us ETC is functioning

    • ETC is functioning but ATP isn’t being produced

      • shows they are linked , but not a direct process

    • If you get the proton across the membrane without going through ATP synthase

      • the delta-G becomes negative again

    • energy gets lost as heat

    • Russians in WW2 took DNP as a way to survive the harsh winter

    • DNP could also be used as a weight loss drug

      • you are not producing ATP , so you are pulling metabolism

        • so eventually you start using fat and glycogen stores to make ATP

      • DNP is very risky , you can kill people with it

        • difference between effective and lethal dose has to be huge

        • DNP however , there differences are very small

          • aka very risk

The F1 Catalyzes

• how does ATP synthase actually make ATP ?

• in the membrane , its oriented in a very specific way

• there’s a proton channel in the portion that’s anchored to the membrane

• the linkage part = the gamma subunit

  • rotates to change the confirmation of the alpha-beta dimers

• there’s 3 alpha-beta dimers , this is where ATP is actually produced

  • these each have 3 different conformations

  • as the gamma rotates , it changes between the 3 confirmations

  • as the protons go through the channel , they rotate the c-subunit

    • they then rotate the gamma

      • as the gamma rotates , it interacts with the alpha-beta dimers to cause conformational change

      • as this happens , the alpha-beta dimers switch between open , loose, and tight

      • every time they switch all the way through , they produce a ATP

      • 1 360 degree rotation produces 3 ATP

        • 1 for each subunit

      • it takes 9 protons to cause a 360 degree rotation

^^ ATP Synthase

^{^} entire process

• we have to move across other things

• for every ATP produced , we have to move 1 additional proton

• so it changes the 9H+ to a 12H+

  • $12H+=3ATP$$12H+ = 3 ATP$

• now the math adds up

• you need 9 protons goin through the ATP synthase channel + 1 proton going through the translocase enzyme

Evidence of Rotation

• covalently attached alpha-beta subunits to a cover slip

• attached actin filament to the gamma subunit

• they saw the actin filament moving

• different organisms have different numbers of c-subunits

  • if you had less c-subunits , you would make more ATP

    • the fewer amount of c-subunits you have the fewer amount of protons necessary to go through the channel

    • if you had 5 , ATP yield would double

    • why don’t humans have 5 then ?

      • less c-subunits requires more force

      • more c-subunits requires less force

      • it might not actually rotate if you get too low on c-subunits

      • if its too high , you make it too easy , you dispel too much of the gradient to make ATP

Malate-Aspartate Shuttle

• NADH that is produced in the cytosolic , has to get to the matrix somehow

  • malate-aspartate shuttle

  • glycerol-3-phosphate shuttle

    • we get less ATP yield with this one

Net Production of ATP

• majority is coming from NADH

  • we need the ETC

• majority is NOT coming from direct substrate level phosphorylation

Regulation of Oxidative Phosphorylation

• majority is being pulled through the respiratory chain

• NADH is a huge inhibitor as we go up the chain

ATP Synthesis Can be Uncoupled

• 1 , 3 , 4 pump protons

• complex 5 pumps it back across

• Uncoupling Protein 1 is used in babies

  • brown adipose tissue is in babies

  • why is it brown ?

    • they have a ton of mitochondria

  • white has mostly fat droplets and only a few mitochondria

  • babies need heat production

    • distribution of brown adipose tissue is all in the trunk , around vital organs

  • uses the proton gradient not for ATP production , but to make heat

Uncouplers of Oxidative Phosphorylation

1. ETC , the higher the amount of uncoupled

   • the higher amount of electron transfer.

   • The regulation isn’t there.

   • P/O ration does down

2. Same thing , we are dispelling the gradient , not producing ATP , dispelled as heat. P/O ratio goes down

3. If P/O ratio gets too low , you don’t make ATP. Cardiac arrest , no brain function

Glucose-6-phosphate

• involved in many different pathways

• in the liver , its a switching system

  • all dependent on need

• Hexokinase IV ( liver ) is special

  • not inhibited by glucose-6-phosphate

• Tissue Specificity :

  • glucose transporters

  • GLUT2 = liver

    • high capacity for glucose

    • glucose can passively diffuse based on concentration

  • GLUT4 = muscle , insulin sensitive version

Metabolism of Amino Acids in the Liver

• centralize around 1 producing the amino acids that need to be released

• also gets rid of the nitrogen

• dumps nitrogen into nitrogen pool , produces aspartate

• glucose-alanine cycle

Fatty Acids (FA) in the Liver

• main fuel source

• liver produces fatty acids

• we take acetyl-CoA to make many things

• what directs metabolism from acetyl-CoA to Ketone bodies ?

  • we don't have oxaloacetate

    • its being pulled off for gluconeogenesis

  • concentration of metabolites

• why in times of fasting are we not making cholesterol ?

  • we don't have the enzymes

  • we shut off the enzymes and degrade them

  • availability of enzymes

Ketone Bodies Are Made from Acetyl-CoA

• $\beta$$\beta$-hydroxybutarate = main ketone body

• brain relies on liver to keep producing ketone bodies

Two Types of Fat Tissue

• Brown expresses the uncoupler protein

  • lots of mitochondria

  • allows ETC to function

  • moves protons into Inner membrane space

  • allows protons to make their way back into the matrix

  • doesn't allow proton gradient to be built

  • bypasses the synthase

  • creates heat

• White is primarily fatty acids

  • very few mitochondria

Muscle

• Difference in amount of mitochondria

• Fast-Twitch have fewer mitochondria

• During light activity , your lipoprotein lipase on the outside of the cell

  • has a really low Km

  • really good at getting fats out of the blood

  • it can also use some ketone bodies and blood glucose

    • blood glucose is insulin dependent

• during heavy activity ,

  • muscle relies on glycogen

  • phosphocreatine helps to produce ATP

Sources of ATP for Skeletal Muscle

• Energy from Phosphocreatine is slightly more thermodynamically favorable

• yields more ATP because you are skipping the hexokinase step

• during glycogen breakdown , how do we guarantee it doesn't leave ?

  • no Glucose-6-phosphatase to remove phosphate from G6P

Hormonal Control of Glycogen Mobilization

• epinephrine and glucagon signal the activity

• whats the difference between epinephrine and glucagon

  • muscle cells don't recognize glucagon , they only respond to epinephrine

• epinephrine activates a bunch of things

  • allows muscle cells to break down glycogen

• with any hormonal signaling , it is amplification

  • 1 epinephrine releases 1000 glucose moieties

  • in hepatocytes , liver responds by exporting more energy into the blood

  • muscle responds with more mobilization

  • small signal , huge response

• people who are fasting ( short-term )

  • start to release epinephrine , helps mobilize energy to get to the next meal

The Cori Cycle

• isn't a closed system

• blood glucose to glycogen , doesn't really enter into muscle cell

• glycogen breaking down glucose

• blood glucose is mostly going to be pulled off into other directions ?

Heart Muscle versus Skeletal Muscle

• very similar in metabolism

• heart has more mitochondria

  • beating all the time

• heart is primarily fueled by fatty acids

  • why ? because it can't store glycogen

    • fatty acids release more energy

  • fatty acids are found in the blood during all stages , fasting , fed , ect

    • so they are always there

    • so the heart always has a source for energy

• if O2 supply cuts off

  • muscle tissue will die

Mammalian Brain

• Fed = glucose

• Starvation = ketone bodies

• If you inject too much insulin ,

  • insulin is an anabolic hormone

  • there are people that use insulin as an anabolic steroid

  • if someone who is not diabetic , and injects too much insulin

    • they can pass out

      • because if you inject too much insulin without eating

        • glucose is taken up by other tissue

        • muscle cells ,

          • there glucose transporter is insulin sensitive

        • the body does not have the time to respond by making ketone bodies

        • the brain doesn't store energy in glucose or ketones

        • its utilizing the glucose as fast as it can bring it in

        • the Km and Vmax are lined up so that glucose is always flowing into the brain at the same rate

          • we use it immediately to make ATP

          • no lag time

        • you pass out , because the brain doesn't have energy

Physiological Effects of Low Blood Glucose

• Kept between a short range , 60 to 90

• If you get too low , you release epinephrine , glucagon , cortosol

• Low glucose levels are a problem

Insulin Stimulates Conversion of Excess Glucose to Glycogen and/or TAGs

• Insulin is the stimulus for converting everything to storage molecules

• Extra acetyl-CoA makes fatty acids --> triacylglycerol --> exported as VLDL

• producing storage molecules based on insulin signal

The Well-Fed Lipogenic Liver

• Intestines have carbs , proteins coming in

  • end up in the liver as glucose

  • glucose-6-phosphate can go to glycogen or pyruvate

FIGURE 23–26 The well-fed state : the lipogenic liver. Immediately after a calorie-rich meal, glucose, fatty acids, and amino acids enter the liver. Insulin released in response to the high blood glucose concentration stimulates glucose uptake by the tissues. Some glucose is exported to the brain for its energy needs, and some to adipose and muscle tissue. In the liver, excess glucose is oxidized to acetyl-CoA, which is used to synthesize fatty acids for export as triacylglycerols in VLDLs to adipose and muscle tissue. The NADPH necessary for lipid synthesis is obtained by oxidation of glucose in the pentose phosphate pathway. Excess amino acids are converted to pyruvate and acetyl-CoA, which are also used for lipid synthesis. Dietary fats move via the lymphatic system, as chylomicrons, from the intestine to muscle and adipose tissues.

Pancreatic Cells

• Pancreas produces the two push or pull hormones

Glucose Regulation of Insulin Secretion

• what causes insulin to be secreated ?

  • glucose transporter into pancreatic beta cell is GLUT2

    • when glucose is above fasting stage , glucose enter beta cells

    • Hexokinase IV is also pancreatic form

    • pancreatic beta cells only import glucose when its high

      • only use when its high

    • after a meal , pancreatic beta cells use glucose

      • they undergo every pathway , to produce ATP

      • when ATP goes high , that inhibits the potassium channel

        • when its inhibited , it causes a depolarization of the membrane

          • stops calcium export

          • causes calcium influx from extracellular space

            • when calcium is high , causes insulin in the secretory granules to release into the cell

              • causes insulin secreation

Sulfonylurea Drugs

• anti-diabetic drugs

• inhibit the potassium channel

• by causing the inhibition , causes more insulin being secreted

  • more insulin secretion

• type 1 diabetes = not making the insulin

  • treatment = add insulin somehow , injections

• type 2 diabetes = insulin insensitive

  • producing insulin , just not recognizing the signal

  • type 2 diabetes drugs help ramp up insulin secreation

    • gets high enough that receptors start to recognize it

The Fasting, Glucogenic Liver

• everything is going towards glucose-6-phosphate

• everything is coming towards the liver , glucose and ketones are going out

• what are the major sources of carbon for gluconeogenesis ?

  • pyruvate

  • alanine = from amino acids

  • glycerol = from adipose tissue

  • NO net conversion of acetyl-CoA to glucose

Glucagon Raises Blood Glucose

• helps keep it at homeostasis

• all of this is coordination

Effects of Prolonged Fasting

• at some point we start to use muscle protein for fuel

  • transamination reactions

• all of the nitrogen ends up as urea

• ketoacidosis , reduces blood pH

  • consequence of diabetes

  • someone who is in a prolonged fast , or keto-diet , why don't they go into ketoacidosis ?

    • getting the fuels , but constantly going toward keto body production

    • everything is going haywire , signaling is going haywire

    • levels are drastically different ?

Fuel Prolonged Fasting or Type 1 Diabetes

• mechanisms are similar

• oxaloacetate is getting pulled off

• doesn't matter type 1 or type 2 ,

  • even if they have high blood glucose , their liver is still producing glucose and ketone bodies

    • not getting the signal to stop

Plasma Levels of Fatty Acids, Glucose, and Ketone Bodies During a One-Week Fast

• fatty acid levels in the blood change during fasting , but relatively constant

• glucose levels after about a month of fasting

• beta-hydroxy-butarate goes up pretty fast

• thats why heart tissue prefers fatty acids over other sources

Triacylglycerol as the majors energy reserve

• Fat vs Carbohydrates

• Fat Advantage = always in the blood circulating

• We have more fat than carbohydrate

• We don’t have to hydrolyze fat , whereas you DO have to hydrolyze carbohydrate

• Glycogen storage has an upper limit

• No upper limit to tryacyl glycerol storage

Primary Sources of TAGs

• Diet

• De novo synthesis

  • make them to use as storage molecules

  • any extra carbohydrate or protein is stored as fat

• Fatty Acid Synthesis Branch Point = Acetyl-CoA

• Muscle , liver , heart takes first dibs , everything else stored in adipose tissue

Digestion of Lipids Overview

• More simpler than carbohydrates

• Starts in the mouth

• Lipases are not caustic ( acidic )

• Proteases are caustic , which is why they are not in the mouth

  • lingual lipase

    • affinity for short or medium chain fatty acids

  • gastric lipase

    • affinity for short or medium chain fatty acids

• Dairy Products have a lot of short and medium chain fatty acids

• Can’t just enter into the intestine , they have to be emulsified into bile salt

• 90% of bile salts get recycled

• Released by cholecystokinin = signaling molecule

• The other difference , is we have to modify the pH via bicarbonate

• Pancreatic Lipase = affinity toward medium and long chain fatty acids.

  • but completely useless without co-lipase

• There are fatty acids attached to other things than triacylglycerols

  • have to be released by other lipases

Lipases Act as Lipid-Water Interface

• The lipases have to work at the lipid-water interface

Phospholipase A₂-Micelle Complex

• Micelles are formed with the lipid inside

• bile salts inhibit the lipase from acting

• Co-Lipase pokes a hole in the exterior

  • allows lipase to interact with the fat

Taurodeoxycholate vs Specific Activity Graph

• Aka lipase activity

• The circles no co-lipase

• Squares = have co-lipase

• Taurodeoxycholate = bile salt

• as we increase concentration of bile salt ,

  • activity of lipase diminishes

• Critical Micelle Concentration

  • the micelles won’t form around the fat unless they reach a certain concentration

  • if they get too high , they also won’t form

  • small window

• they are active until micelles forms

  • once they form , there is no activity

  • unless you add co-lipase , then activity resumes

Bile Salts

• Base = cholesterol

• 7-alpha-hydroylase = rate limiting step

  • inhibited by bile acids

• just help with surface area and emulsified

• if you don’t have bile salts , your digestive tract can’t digest lipids

  • leads to weight loss and gastric stress

• some bile salts are more hydrophobic or hydrophilic than others

• 95% of Bile salts are reabsorbed in gallbladder

Olestra

• Weight loss drug

• added to food

• pancreatic lipase can’t break it down ( it can’t bind )

• it tastes like fat , but intestinal tract can’t break it down

• they include fat soluble vitamins

  • people who eat a ton of these foods become vitamin D deficient

  • Vitamin D is fat soluble

    • gets removed along with drug

  • so they “fortify” these foods with extra vitamins

Orlistat

• Compound that inhibits pancreatic lipase

• dosing has to be really fine-tuned

  • have to restrict all fat in diet

Processing of Dietary Lipids

• In the intestine , pancreatic lipase takes triacyl glycerol and breaks it down into 2 fatty acids plus a mono-acyl-glycerol

• The 2 FA and MAG enter

• we have no mechanism to get triacyl glycerol into dig

• we reform them , because we don’t want to send out that much fat into the blood

• so we take that triacyl glycerol and package it as chylomicrons

• Chylomicrons = lipoprotein

  • on the outside its similar to micelle , but they have apoproteins

    • apoprotiens help with structure and solubility

• FA could dissociate out ,

  • so we convert them back into TAG

• Apoproteins are recognized by surface molecules on muscle cells and help direct them to their target

Chylomicrons

• Made in intestinal cells

• Starting point for lipoproteins = chylomicrons or VLDL ( very-low-density-lipoprotein )

• Chylomicrons = exogenous , dietary fats

  • formed in the intestine

  • protein portion is made in rough ER

  • triacyl-glycerol is reformed once inside enterocytes

• VLDL = fats we make from carbohydrate of protein

  • made in the liver

• Picks up additional lipoproteins

  • E = helps recognized by the liver

  • C2 = helps activate lipoprotein lipase

• Chylomicron with lots of triacyl glycerols in it

  • Apoproteins on the outside of Chylomicron

  • Cell Surface of muscle and adipose is lipoprotein lipase

  • C2 = goes activate

    • triacyl-glycerols are then broken down , and then enter into cell

• Difference between lipoprotein lipase in muscle and in adipose tissue

  • Muscle has a very low Km

  • adipose has a medium Km , orders of magnitude different

  • Muscle obviously uses Fatty acids first before we start storing it.

    • It also stays active longer as fatty acids start to dissipate in the blood

• Once the lipoprotein lipase on adipose tissue and muscles are being utilized , the fats get removed

• Chylomicrons = even lower density than VLDL

• as we remove the fats , the proteins stay , the density increases

• Chylomicron Remenents are more dense

  • they go to the liver

  • apoE = gets recognized , further digested

Four Major Classes of Lipoprotein Particles

• VLDL = fats from what we are producing from dietary source of carbohydrates and protein

• Chylomicrons have very little protein , etc , but a ton of tryacyl glycerols

• when we get to VLDL , they have more

  • the liver is packaging all of this up to be transported out

• Free cholesterol vs cholesterol esters

  • cholesterol esters have a fatty acid esterified to them

    • much more hydrophobic

    • likely to stay in the core of the lipoprotein

  • free cholesterol is less likely to stay in the lipoprotein

• How do we go from LDL to HDL ?

  • Liver produces VLDL

  • VLDL goes off to muscle and adipose

  • VLDL interacts with lipoprotein lipase on surface

    • removes fatty acids

    • produces IDL

  • IDL is somewhere between VLDL and LDL in its density

  • 50% of IDL goes back to the liver

  • The other 50% goes back to another round with interaction on muscle and adipose tissue

    • this produces LDL

  • the amount of triaclyglycerides keeps decreasing

    • we are removing the fats

    • the amount isn’t higher , the percentage is higher

  • the majority of LDL goes to the liver

  • LDL = “bad” , because some cholesterol goes to peripheral tissues

    • but we have a limited capacity to process LDL

    • if you are eating too much fats , LDL percentage goes up , you have a limited capacity to uptake it

      • the more its circulating in the blood , the more likely it is to be oxidized

        • oxidized LDL starts to form plaques / lesions

    • old school limiting lowering drugs targeted how much LDL we have

    • newer ones target liver and its capacity to take up LDL

   

Apolipoproteins in Lipoproteins

• apo = protein protion

• B48 = for chylomicrons

• B100 = same function as B48

  • transcribed form the same gene

  • b48 has a stop codon that gets put in , and makes only about 48%

  • VLDL expresses B100

  • Chylomicrons express B48

Electron Microscope Pictures of Lipoproteins

• Chylomicrons = much bigger

• VLDL smaller

• keeps going down in size

Biological Roles

• HDL = responsible for reverse cholesterol transport

  • cholesterol back to the liver

    • why its considered the “good” cholesterol

  • can be made in the blood

    • if we have the apo-proteins

• these are the 4 main particles

• changing the density comes from lipoprotein lipase activity

  • removing the fats from it

Cholesterol

• HDL = takes cholesterol back to the liver

  • has a lot of cholesterol esters in it

    • cholesterol esters are more hydrophobic than cholesterol itself

    • there are two enzymes that make cholesterol esters = ACAT and LCAT , esterify

    • Helps keep cholesterol in the HDL molecule

  • hydrophobic because of the long R-group

• VLDL has a lot of triacylglycerols around

  • cholesterol ester transfer protein

    • takes triacylglycerols from the VLDL and moves them to the HDL

    • it also moves cholesterol esters into the VLDL

    • makes HDL with a low concentration of esters

      • makes it less cardio protective protective

     

Cholesterol Metabolism

• Synthesis = complicated process with black box enzymes

  • just know that all of the carbon can come from acetyl-CoA or acetate

    • goes to mevalonate —> isoprenes

  • rate limiting step is HMG-CoA synthase / reductase

    • also happens to be the enzyme we target with medications

  • eventually we form squalene , its the linear form of cholesterol

  • ultimately we form the steroid ring in multiple steps

• Regulation :

  • 5 different ways we regulate synthesis

  • 1 = regulate function of regulation step ( activity )

    • via phosphorylation / dephosphorylation

  • 2 = regulate amount

    • turn it off short term , degrade it if we don’t need it again

    • produce it long term if we need it

  • Insulin activates HMG-CoA reductase

    • insulin indirectly regulates through phosphorylation cascade

  • Glucagon is the opposite

  • Regulate through short term via phosphorylation / dephosphorylation

  • Long Term Regulation :

    • Transcription level control

      • normally , we have several things in the membrane; : Insig , SCAP are all in the membrane when cholesterol / sterols levels are high

      • they bind to the proteins and keep them associated

      • when cholesterol levels lower , SCAP and Insig separate , causes a conformational change , allows for proteases to cleave off a transcription factor from SREBP

        • part of SREBP is a transcription factor

• HMG-CoA reductase when cholesterol levels go up

  • there are portions that are sterol sensing

    • when they start to bind the cholesterol , it causes conformational change and changes how the protein interacts

      • its now a target for proteolytic cleavage

  • when there’s no cholesterol its in the active form and functioning

• 2 Long term regulation :

  • when low = transcription level activation

  • when its high = we are getting rid of the protein concentration itself ( increasing proteolytic cleavage )

Regulation of HMG-CoA Reductase

• LXR = transcription factor

• if cholesterol levels are high , it binds to the transcription factor , changes state , allows for production of enzymes

• when low , LXR binds to the repress and stops production of genes

• Acetyl-CoA carboxylase = fatty acid synthesis

• ABCA1 , ABCG1 = reverse cholesterol transport

• others are involved in lipid synthesis

Lipoprotein Receptor

• can recognize ApoE or ApoB100

  • when binds , it internalizes via receptor mediated endocytosis

  • 1 in 500 have a defect in this receptor

    • you can’t uptake any lipoproteins , much slower at it

      • increases concentration in the blood

      • becomes oxidized , leads to atherosclerosischlerosis

Atherosclerosis

• its the amount of oxidized LDL binding to other things floating around and forming plaques

• forms foam cells

Drug Therapies

• statins are the gold standard

  • research suggests it lowers cardiac diseases

  • its also very cheap , off-patent

  • they inhibit HMG-CoA reductase

    • limits self-synthesis of cholesterol

      • doesn’t help with any dietary intake

• Bile Salt and Resins :

  • bile salts are made from cholesterol

    • typically 95% of bile salts are recycled

    • if you bind bile salts and secrete them ,

      • more cholesterol is delegated to re-forming bile salts

    • resin = surface for bile salt to bind

• Naicin and Fibrates are Second line defense

• Ezetimibe = reduces intestinal absorption in the gut

  • limits a transporter

HMG-CoA Reductase Inhibitors

• aka statins

• mostly competitive inhibitors

• red shape of Lipitor basically matches the shape of HMG-CoA

  • binds to HMG-CoA reductase , and inhibits it

• Disadvantage of Competitive Inhibitors :

  • upper threshold you can’t give past a certain point

• Advantage of Competitive Inhibitor vs Co-Valent Inhibitor :

  • covalent inhibitor lasts so much longer

  • aspirin is short acting , 1 and done

• Aspirin vs Ibeprophan :

  • aspirin is covalently inhibitor

    • binds to COX and inhibits it

Repatha

• PCSK9 inhibitors

• newest drug

• in the internalization and degradation of the receptor , there is an additional protein involved

• if we don’t degrade the receptor ,

  • we have more exceptions expressed ,

• By having more receptors present , even at a reduced capacity , it’s still capable

• Disadvantage = monoclonal antibody

  • expensive , has to be injected

  • reserved for people without cardiac disease

HDL

• formation of cholesterol ester is really important

• LCAT vs ACAT

Reverse Cholesterol Transport

• HDL also can help mature lipoprotein

• TAG vs CE

• modifying how much is going back to the liver , and how much is being processed

Questions

1. What is the approximate number of protons that are transported across the mitochondrial membrane to produce 3 ATP?

   • 12

   • It takes 12 protons to transport across the membrane,

   • 9 for a complete 360 degree rotation of the gamma subunit and 3 for the phosphate transport

2. Oxidative phosphorylation is mainly regulation by which of the following?

   • Levels of NADH and ADP

   • Ox/Phos is mainly regulated by substrate availability

3. What is the main energy source for heart tissue?

   • Fatty Acid

   • Heart tissue has a high energy demand at all times.

     • which makes fatty acid the one fuel that would be available in both the fed and fasting state

4. High levels of cholesterol will alter cholesterol metabolism in which of the following ways :

   • Oligomerize HMG-CoA reductase increasing proteolytic cleavage

   • HMG-CoA has a sterol sensing domain, as cholesterol levels increase it causes the enzyme to oligomerize and degrade

5. Which of the following is responsible for transporting fat from dietary sources :

   • Chylomicrons

   • Chylomicrons are dietary fats

     • Whereas VLDL are endogenous fats

High-Level Overview

• Glycerolneogenesis vs gluconeogenisis ?

• Adipose tissue doesn't have glycerol kinase ?

• cholesterol metabolism ( study a lot )

  • regulation

    • HMG-CoA reductase = rate limiting step

      • regulated via :

        • insulin signaling

        • phosph / dephosph

        • transcription levels ,

        • proteolytic cleavage = conformatonal change

          • as sterols in cell go higher , oligiomerzation state changes

            • leaves it susceptible to cleavage

  • production of VLDL , HDL

    • VLDL is produced in the liver

      • transported via blood

        • ..... lots more of the story

          • eventually get to LDL

            • to LDL receptor on liver

              • endocytose , ......

• urea cycle ( also study )

  • regulated

    • never turn it off

    • its a feed-forward reaction

    • turn it up in times of need

      • high nitrogen turn off

        • turns on CPS-1 ?

  • amino-acid transport ( nitrogen transport )

    • key sources of nitrogen

    • transported via ammonium ( $N{H}_4^{+}$$NH_4^+$ ) and aspartate

      • glucose alanine shuttle

        • Liver and muscle

          • transamination pairs

• oxidatative phosphorylation ( easier )

  • How do we go from NADH to ATP

    • indirect

      • Not substate level

      • atp synthase complex 5

      • substrate level = pyruvate kinase ?

  • regulation of ETC

    • when ATP is high , we stop producing it via complex 5

      • delta g becomse positive , electron transport stops

        • increases NADH

          • inhibits everything above it

• uncouplers

  • uncoupels ETC and oxidative phosphorylation

  • if you add an uncoupler to mitochondria

    • ETC skyrockets

    • oxidative phosphorylation stops

Insulin mediated signaling

• Receptor :

  • type of tyrosine kinase

  • Alpha subunit :

    • localized in extracellular region

    • also has a transmembrane domain

    • contains the ligand binding site

  • Beta Subunit :

    • involved in intracellular signaling

• Insulin binds

  • causes monomers to dimerize

    • tyrosine kinase regions get phosphorylated

      • causes the activation loop to change confirmation

        • opens up substrate binding site for further cellular responses / signaling

• Phosphorylated tyrosine residues act as a docking site for SHC and IRC ( adaptor proteins )

• Phosphorylation of the intracellular tyrosine kinase can create two signals

  • Shc activation = signals growth

    • via Grb2 recruitment

      • starts the MAPK signaling pathway

        • enhances cell growth

  • IRS activation = signals metabolism

    • "insulin receptor substrates" ( IRS )

      • starts the PI3-kinase signaling pathway

        • PIP2 ➡️ PIP3 ➡️ activates Protein Kinase B ( Akt )

          • regulates glucose homeostasis and a bunch of other things

            • aka glucose import into the cell and glycogen synthesis

• There are also serine phosphorylation sites on IRS proteins

  • these allow for modulation of insulin signaling

    • positive / negative feedback

  • prolonged activation of serine residues leads to insulin resistance

Insulin Resistance

• defined as normal or elevated insulin causing an attenuated biological response

  • aka , a decreased biological response to normal concentrations of circulating insulin

    • aka insulin insensitivity

• If those serine sites are phosphorylated on IRS proteins :

  • the IRS can't attract PI3K anymore

  • effectively , it decreases IRS-1 tyrosine phosphorylation

    • impairs its downstream targets

• circulating FFA and adipokine TNF𝛼 can increase serine phosphorylation

What is metabolic syndrome

• metabolic syndrome = inability to store triacylglycerides

• its not a disease , but rather a cluster of metabolism disorders

  • high blood pressure

  • high insulin levels

  • excess body weight

  • abnormal cholesterol levels

• Excessive lipids are stored in abnormal ( ectopic ) places

  • problem / toxic

• Individuals with metabolic syndrome tend to develop type 2 diabetes

  • MCP-1 recruited macrophages

  • if muscle can still store fats , it’s somewhat ok.

  • only if fat is being stored in like the liver or elsewhere do we consider it type II diabetes

• In obesity , the capacity of adipocytes to store TAGs becomes exhausted & several changes occur :

  • Adipocytes become less sensitive to insulin

  • Gene expression associated with the development of new adipocytes is down-regulated

  • Expression of transcription factors in liver and skeletal muscle upregulates the lipid synthesizing machinery and increases lipid storage capacity

Drugs that increase insulin sensitivity or insulin production

• Sulfonylureas :

  • examples = Glyburide , glipizide , and glimepiride

  • Blocks ATP sensitive K-channels

    • causes depolarization and opening of voltage-gated calcium channels

      • increases intracellular calcium in the beta cells

        • stimulates insulin release

• Biguanides :

  • example = Metformin

  • inhibits mitochondria complex 1

    • inhibits cAMP formation , aka causes high AMP concentration

      • activates AMPK

        • shifts metabolism to conserve energy

        • decrease in lipogenic gene expression

          • aka increase :

            • glucose uptake and oxidation

            • increase fatty acid mobilization and oxidation

            • inhibits synthesis of glucose , FA , and sterols

• Thiazolidinediones ( TZDs ) :

  • examples = troglitazone , rosiglitazone , and pioglitazone

  • TZDs bind avidly to peroxisome proliferator-activated receptor gamma ( PPAR𝛾 ) in adipocytes

    • promotes adipogenesis and fatty acid uptake

• Glucagon-Like Peptide-1 ( GLP-1 ) Agonists :

  • examples = Exenatide , Sitagliptin , and Ozempic !

  • GLP-1 binds to receptors on beta cells

    • activates adenyl cyclase to form cAMP

      • cAMP activates PKA and Epac2

        • increases intracellular calcium

          • promotes insulin secretion

Summary

• The insulin receptor, INSR, is the prototype of receptor enzymes with Tyr kinase activity. When insulin binds, each αβ unit of INSR phosphorylates the β subunit of its partner, activating the receptor’s Tyr kinase activity.

  • The kinase catalyzes the phosphorylation of Tyr residues on other proteins, such as IRS-1.

• Phosphotyrosine residues in IRS-1 serve as binding sites for proteins with SH2 domains.

  • Some of these proteins, such as Grb2, have two or more protein-binding domains and can serve as adaptors that bring two proteins into proximity.

• Sos bound to Grb2 catalyzes GDP-GTP exchange on Ras (a small G protein), which in turn activates a MAPK cascade that ends with the phosphorylation of target proteins in the cytosol and nucleus.

  • The result is specific metabolic changes and altered gene expression.

• The enzyme PI3K, activated by interaction with IRS-1, converts the membrane lipid PIP2 to PIP3

  • which becomes the point of nucleation for proteins in a second and third branch of insulin signaling.

• Metabolic syndrome, which includes obesity, hypertension, elevated blood lipids, and insulin resistance, is often the prelude to type 2 diabetes.

• The insulin resistance that characterizes type 2 diabetes may be a consequence of abnormal lipid storage in muscle and liver, in response to a lipid intake that cannot be accommodated by adipose tissue.

• Effective treatments for type 2 diabetes include exercise, appropriate diet, and drugs that increase insulin sensitivity or insulin production.

  • Surgical alteration of the digestive tract leads to weight loss and often reverses type 2 diabetes.

Lipid Storage

• Double bonds create kinks

  • reduces the ability to compact

• lipids are reduced compounds

  • lots of potential energy

• hydrophobic in nature = good for packing

• lipids are usually stored as triacylglycerols

• most natural fatty acids :

  • between 12 and 18 carbons in length

  • even numbered

  • unbranched

• chain length $\frac{1}{\propto }$$\frac{1}{\propto}$ solubility

• chain length $\propto$$\propto$ melting temperature

• number of double bonds $\frac{1}{\propto }$$\frac{1}{\propto}$ melting temperature

  • kinks are the enemy of compaction

  • to fight against becoming a solid , we introduce kinks

    • aka double bonds

• Poly Unsaturated :

  • liquid at room temperature

  • melting temp $\propto$$\propto$ number of double bonds

• Trans fatty acids can pack more tightly than cis fatty acids

  • therefore , they have a higher melting temperature than cis

• Triacylglycerols ( simplest form of a lipid ) are less soluble in water than free floating fatty acids

  • due to the esterification of the carboxylate group

• Lipids are water-insoluble cellular components, of diverse structure, that can be extracted from tissues by nonpolar solvents.

• Almost all fatty acids, the hydrocarbon components of many lipids, have an even number of carbon atoms ( usually 12 to 24 )

  • they are either saturated or unsaturated, with double bonds almost always in the cis configuration.

• Triacylglycerols contain three fatty acid molecules esterified to the three hydroxyl groups of glycerol

  • simple triacylglycerols contain only one type of fatty acid

  • mixed triacylglycerols contain two or three types

• Triacylglycerols are primarily storage fats; they are present in many foods.

• Because trans fatty acids in the diet are an important risk factor for coronary heart disease, their use in prepared and processed foods has become highly regulated.

• Waxes are esters of long-chain fatty acids and long-chain alcohols.

Lipid Structures

• Glycerophospholipids :

  • main lipid type in cell membrane

    • mostly phosphatidylcholine

  • also found in mitochondrial membrane

  • have different head groups

  • they can donate protons

    • aka carry a negative charge

    • allows them to recruit proteins to bind to the lipid

• Ether Lipids :

  • Plasmalogen :

    • common in heart tissue

    • vinyl ether analog of phosphatidylethanolamine

  • Platelets-Activating Factor :

    • aliphatic ether analog of phosphatidylcholine

    • first signaling lipid to be identified

• Sphingolipids :

  • backbone is NOT a glycerol

    • its a long chain amino alcohol called a sphingosine

  • fatty acid is joined via an amide linkage instead of the usual ester bond

  • found mainly in the outer face ? of the plasma membrane

  • ceramide is the base sphingolipid , used to construct others

  • sphingomyelin = type found in myelin sheaths

    • basically the same thing as phosphatidylcholine , except it has the sphingosine backbone instead of a glycerol

  • blood groups are determined in part by the type of sugars on the head groups in glycosphingolipids

    • sugar structure is determined by which glycosyltransferase is expressed

      • no glycosyltransferase expressed = O group

      • one that transfers N-acetylgalactosamine = A group

      • one that transfers a galactose = B group

• Sterols and Cholesterols :

  • steroid nucleus = 4 fused rings

    • the fused rings make it almost planer ( basically rigid )

  • modulate membrane fluidity and permeability

  • thicken the plasma membrane

  • most bacteria do NOT have sterol / cholesterol

  • mammals get cholesterol from diet , or the liver can synthesize it

  • cholesterol is transported to tissues via blood stream

    • must be bound to a protein

  • most hormones are derivatives of sterols

  • steroids :

    • oxidized derivatives of sterols

    • have the sterol nucleus , but don't have the alkyl chain like cholesterol does

    • more polar than cholesterol

    • steroid hormones are synthezied in the gonads or adrenal glands from a cholesterol base

• The polar lipids, with polar heads and nonpolar tails, are major components of membranes. The most abundant are the glycerophospholipids, which contain fatty acids esterified to two of the hydroxyl groups of glycerol, and a second alcohol, the head group, esterified to the third hydroxyl of glycerol via a phosphodiester bond. Other polar lipids are the sterols.

• Glycerophospholipids differ in the structure of their head group; common glycerophospholipids are phosphatidylethanolamine and phosphatidylcholine. The polar heads of the glycerophospholipids are charged at pH near 7.

• Some archaea have unique membrane lipids, with long-chain alkyl groups ether-linked to glycerol at each end and with sugar residues and/or phosphate joined to the glycerol to provide a polar or charged head group. These lipids are stable under the harsh conditions in which these archaea live.

• The sphingolipids contain sphingosine, a long-chain aliphatic amino alcohol, but no glycerol. Sphingomyelin has, in addition to phosphoric acid and choline, two long hydrocarbon chains, one contributed by a fatty acid and the other by sphingosine. Three other classes of sphingolipids are cerebrosides, and gangliosides, which contain sugar components.

• The Sterols have four fused rings and a hydroxyl group. Cholesterol, the major sterol in animals, is both a structural component of membranes and precursor to a wide variety of steroids.

Fat-Soluble Vitamins

• K - A - D - E

• K :

  • cofactor for a carboxylase in the coagulation pathway

  • if deficient , it leads to bleeding problems

  • warfarin blocks this pathway

• A :

  • Involved in visual pigment

    • isoprene units allow electron delocalization / movement

  • Precursor for other hormones involved in signaling

• D :

  • forms in the skin by a reaction driven by sunlight; in the liver/kidney

  • converted to a biologically-active hormone that regulates calcium uptake

• E :

  • antioxidant

Arachidonic Acid ( AA ) Derivatives

• Phospholipases can breakdown / cleave different membrane phospholipids into Arachidonic Acid + LPC

• Arachidonic Acid can go through different enzymes to turn into different derivatives

  • AA + COX = Prostaglandin , prostacyclin , thromboxane

  • AA + LOX = Leukotrienes , HETEs , HPETEs

  • AA + CYP2C = EETs

• Prostaglandins = anti-inflammatory , anti-fever

• Thromboxanes = form blood clots

• Leukotrienes = smooth muscle contraction in lungs

Fatty Acid Nomenclature

• Delta vs Omega Nomenclature

• Common Names :

  • Palmitic Acid ( 16:0 )

  • Stearic Acid ( 18:0 )

  • Palmitoleic Acid ( 16:1n-9 )

  • Oleic Acid ( 18:1n-9 )

  • Linoleic Acid ( 18:2n-6 )

  • $\alpha$$\alpha$-Linolenic Acid ( 18:3n-3 )

  • Arachidonic Acid

  • EPA

  • DHA

Essential Fatty Acids

• NOT produced in the human body

  • must be obtained in diet

• Linoleic Acid , aka Omega-6 = $18:2\left({\mathrm{\Delta }}^{9,12}\right)$$18:2\left(\ \Delta^{9,12} \ \right)$

  • base for making arachidonic acid

    • eicosanoids

• $\alpha$$\alpha$-Linolenic Acid , aka Omega-3 = $18:3\left({\mathrm{\Delta }}^{9,12,15}\right)$$18:3\left(\ \Delta^{9,12,15} \ \right)$

  • base for making eicosapentaenoic acid ( EPA ) and docosahexaenoic acid ( DHA )

    • anti-inflammatory

Summary

• Some types of lipids, although present in relatively small quantities, play critical roles as cofactors or signals.

• Phosphatidylinositol bisphosphate is hydrolyzed to yield two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate is a nucleation point for supramolecular protein complexes involved in biological signaling.

• Prostaglandins, thromboxanes, leukotrienes, and lipoxins, all of which are eicosanoids derived from arachidonate, are extremely potent hormones.

• Steroid hormones, such as the sex hormones, are derived from sterols. They serve as powerful biological signals, altering gene expression in target cells.

• Vitamins D, A, E, and K are fat-soluble compounds made up of isoprene units. All play essential roles in the metabolism or physiology of animals. Vitamin D is precursor to a hormone that regulates calcium metabolism. Vitamin A furnishes the visual pigment of the vertebrate eye and is a regulator of gene expression during epithelial cell growth. Vitamin E functions in the protection of membrane lipids from oxidative damage, and vitamin K is essential in the blood-clotting process.

• Lipidic conjugated dienes serve as pigments in flowers and fruits and give bird feathers their striking colors.

• Polyketides are natural products widely used in medicine.

Reagents to Extract Neutral and Membrane Lipids

• Neutral ( TG , Waxes ) :

  • use hydrophobic solvents

  • Ethyl ether , chloroforms , benzene

• Membrane :

  • use more polar solvents

  • Ethanol , methanol

Basic Principles of TLC , GC , HPLC , MS

• TLC :

  • involves a stationary phase (usually a thin layer of adsorbent material like silica gel or alumina coated onto a glass or plastic plate) and a mobile phase (solvent or solvent mixture). A small amount of sample is applied near the bottom of the plate, which is then placed vertically in a developing chamber containing the mobile phase. As the mobile phase moves up the plate, different compounds in the sample migrate at different rates due to their varying interactions with the stationary phase. The separation is visualized as spots on the plate, and the retention factor (Rf) is used to compare and identify compounds.

• GC :

  • GC is a separation technique used for volatile compounds. It consists of a stationary phase (a solid support with a liquid or solid coating) and a mobile phase (an inert gas like helium, nitrogen, or hydrogen). The sample is vaporized and carried through a long column by the mobile phase. Compounds in the sample interact with the stationary phase and are separated based on their differing boiling points and affinity for the stationary phase. As the compounds exit the column, they are detected by a detector (e.g., flame ionization detector or mass spectrometer), and the retention time is used to identify and quantify the compounds.

• HPLC :

  • HPLC is a widely used separation technique for a broad range of compounds. It uses high pressure to force the mobile phase (a liquid solvent or solvent mixture) through a column packed with a solid stationary phase (e.g., silica or polymer-based particles). The sample is injected into the mobile phase, and compounds in the sample interact with the stationary phase, resulting in separation based on their polarity, size, or other properties. The compounds are detected as they elute from the column, and the retention time is used for identification and quantification. Different types of HPLC include reversed-phase, normal-phase, size-exclusion, and ion-exchange chromatography.

• MS :

  • MS is an analytical technique used to identify and quantify compounds based on their mass-to-charge ratio (m/z). The sample is ionized by an ionization source (e.g., electron impact or electrospray), and the ions are separated by a mass analyzer (e.g., quadrupole, time-of-flight, or orbitrap) according to their m/z. The ions are then detected, and the resulting mass spectrum provides information about the molecular mass, elemental composition, and structure of the compounds. MS can be used as a standalone technique or coupled with other separation methods like GC or HPLC for more comprehensive analysis.

Summary

• In the determination of lipid composition, the lipids are first extracted from tissues with organic solvents and separated by thin-layer, gas, or high-performance liquid chromatography.

• Phospholipases specific for one of the bonds in a phospholipid can be used to generate simpler compounds for subsequent analysis.

• Individual lipids are identified by their chromatographic behavior, their susceptibility to hydrolysis by specific enzymes, or mass spectrometry.

• High-resolution mass spectrometry allows the analysis of crude mixtures of lipids without prefractionation—the “shotgun” approach.

• Lipidomics combines powerful analytical techniques to determine the full complement of lipids in a cell or tissue (the lipidome) and to assemble annotated databases that allow comparisons between lipids of different cell types and under different conditions.

Questions

1. The action of metformin is to increase ATP production ?

   • False

2. The glycerophospholipids are composed of two fatty acids attached to glycerol and threonine ?

   • False

3. The main difference between saturated and unsaturated fatty acids is :

   • the presence of double bonds

4. How do free fatty acids contribute to impaired insulin signaling response ?

   • Free fatty acids activate IRS-1 serine phosphorylation

5. Name the two essential fatty acids

   • Linoleate and Linolenate

6. The melting point of fatty acids depends upon chain length and _______ ?

   • Degree of unsaturation

Enzymes involved in Synthesis

• Acetyl-CoA Carboxylase :

  • Bi-Functional enzyme with 3 active domains

    • Biotin carrier protein = swing arm

      • transfers activated CO2 from biotin carboxylase domain to the transcarboxylase active site

    • Biotin Carboxylase = carboxylates biotin

    • Transcarboxylase = transfers activated CO2 from biotin to acetyl-CoA , forming malonyl-CoA

• Fatty Acid Synthase :

  • 2 major isoforms

  • FAS 1 = found in vertebrates and fungi

    • single multifunctional polypeptide chain that catalyzes 7 different reactions

      • a dimer of two identical monomers

      • each monomer has 7 active sites in 7 different domains :

        • 5 of those are actual catalysis enzymes

        • 1 is an acyl carrier protein ( ACP ) = swing arm

          • Flexible arm to carry intermediates from one enzyme subunit to the next

        • 1 is a thioesterase that releases the palmitate product from ACP

  • FAS 2 = found in plants and bacteria

  • Advantages of Multi-Enzyme Complex :

    • active sites are all centralized

      • higher efficiency

    • all 7 enzymes are encoded by a single gene

Reactions for Fatty Acid Synthesis

• acetyl and malonyl group activation

  • acetyl group of acetyl-CoA is first transferred to the –SH group of the b-ketoacyl-ACP synthase (KS)

    • in a reaction catalyzed by acetyl-CoAACP transacetylase (AT)

  • The malonyl group is transferred from malonyl-CoA to the –SH group of acyl carrier protein (ACP)

• 4 step elongation reaction

  • Acetyl-CoA is the priming group only in the first cycle

    • after that, only malonyl-CoA is added to the ACP each time

  • After activation , there is a four-step process

  1. Condensation : of the growing chain with activated acetate

  2. Reduction : of carbonyl to hydroxyl

  3. Dehydration : of alcohol to trans-alkene

  4. Reduction : of alkene to alkane

  • Each cycle results in the net addition of two carbons to the growing fatty acid chain.

  • These steps are repeated till a fatty acid with 16 carbon atoms is synthesized.

• termination reaction

  • Seven rounds of the four-step lengthening reactions produces palmitoyl-ACP

  • Palmitoyl-ACP is hydrolyzed by a thioesterase to release a free palmitate

  • This is why it there are only 6 net H2O molecules.

    • one gets "burnt" here to hydrolyze free palmitate

Why are there only 6 net $H_{2}O$$H_2O$ molecules in the synthesis?

• Overall, the biosynthesis of FA requires acetyl-CoA and the input of energy in the form of ATP and reducing power of NADPH.

• $8\mathrm{acetyl}-\mathrm{CoA}+7\mathrm{ATP}+14\mathrm{NADPH}+14\mathrm{H}{\phantom{A}}^{+}⟶\mathrm{Palmitate}+7\mathrm{ADP}+7\mathrm{Pi}+8\mathrm{CoA}+14\mathrm{NADP}++6\mathrm{H}{\phantom{A}}_2\mathrm{O}$$\ce{8acetyl-CoA + 7ATP + 14 NADPH + 14H+ -> Palmitate + 7ADP + 7Pi + 8CoA + 14NADP + + 6H2O}$

• What is the Function of ATP ?

  • used to synthesize malonyl-CoA

• What is the function of NADPH ?

  • used in the 2nd and 4th steps as the electron donor

Locations of Fatty Acid Synthesis

• acetyl-CoA Shuttle :

  • citrate shuttle on inner mitochondrial membrane

    • shuttles acetyl-CoA out of the membrane as citrate

  • acetyl-CoA is made from glucose and amino acids in mitochondria

  • Intramitochondrial acetyl-CoA first reacts with oxaloacetate to form citrate

    • in the TCA cycle catalyzed by citrate synthase.

  • Citrate then passes into the cytosol through the mitochondrial inner membrane on the citrate transporter

  • Acetyl-CoA is regenerated by the action of ATP-dependent citrate lyase in the cytosol

  • Oxaloacetate is shuttled back into the mitochondria as malate or pyruvate

• Elongation = mitochondria

• Desaturation = endoplasmic reticulum

Factors Involved in the Process of Fatty Acid Synthesis

• NADPH is used as a reducing agent / electron donor

  • synthesized in :

    • pentose phosphate pathway

    • from malic enzyme

Regulation of Fatty Acid Synthesis

• what is the rate limiting step?

  • converting the phosphorylated / inactive version of the promotors into the dephosphorylated / active / polymer form

  • Acetyl-CoA carboxylase regulates rate-limiting step of fatty acid synthesis

• How is acetyl-CoA carboxylase regulated ?

  • ACC is regulated by phosphorylation/dephosphorylation and protein polymerization

  • ACC forms long , active filamentous polymers from inactive protomers

  • Palmitoyl-CoA = allosteric inhibitor ( feedback inhibition )

  • Citrate = allosteric activator ( ATP and acetyl-CoA high in mitochondria )

  • When we have enough FA ( palmitoyl-CoA ) , we inhibit malonyl-CoA

• Insulin signaling leads to dephosphorylation of ACC

  • activates ACC activity to ensure that excess glucose will be rapidly converted to fatty acid for long term energy storage

• Glucagon and epinephrine triggers phosphorylation

  • inactivates acetyl CoA carboxylase

Fatty Acid Elongation and Desaturation

• Understand the location

  • palmitate ( C16 - saturated ) is the final product

    • anything longer or anything with double bonds has to be transported to the smooth E.R.

• Understand electron transfer

  • The double bonds are introduced by the catalysis of fatty acyl-CoA desaturase (a mixed-function oxidase)

    • where both the fatty acyl group and NADPH are oxidized by O2 .

  • The electrons of NADPH are transferred to O2 via Cyt b5 reductase and cytochrome b5

Prostanoid Biosynthesis

• COX is an enzyme that catalyzes the rate determining step in the conversion of AA to prostanoids by a 2-step process involving oxygen.

• Cyclooxygenase adds two molecules of O2 to AA to form the endoperoxide PGG2

• PGH2 serves as a “branch point” for specific enzymes leading to the formation of prostacyclin, prostaglandins, and thromboxanes.

NSAIDs action of Mechanism

• Aspirin inhibits the first reaction by acetylating an essential Ser residue on the enzyme.

• Ibuprofen and naproxen inhibit the same step, probably by mimicking the structure of the substrate or an intermediate in the reaction.

• Slide 40 on Lecture 02 and 03

The Precursor

• Phosphatidic Acid is the common precursor for the synthesis of both triglycerides and glycerophospholipids

PA synthesis and Conversion of PA to TG

• Phosphatidic acid is made by transferring two acyl groups from two acyl-CoAs to L-glycerol 3-phosphate

  • L-glycerol 3-phosphate is derived from either glycerol or dihydroxyacetone phosphate

• A phosphatidic acid is converted to a triglyceride via a dephosphorylation reaction

  • catalyzed by phosphatidic acid phosphatase and an acyl transferring reaction

• The vast majority of the glycerol 3-phosphate is derived from the glycolytic intermediate dihydroxyacetone phosphate (DHAP)

• In liver, a small amount of glycerol 3-phosphate is also formed from glycerol by the action of glycerol kinase.

• Fatty acyl-CoA synthetase (ACS) catalyzes the ATP-dependent formation of a thioester bond between a fatty acid and coenzyme A.

• One mole of L-glycerophosphate acylated with 2 mol of CoA–fatty acids yields PA.

Re-Esterification and Action of Mechanisms of Dexamethasone and Thiazolidinediones

• Glucocorticoid hormones stimulate glyceroneogenesis in the liver

  • while suppressing glyceroneogenesis in adipose tissue which is controlled by the activity of PEPCK

• Dexamethasone = corticosteroid

  • in adipose tissue :

    • decreases transcription of PEPCK

      • inhibits glyceroneogenesis

  • in liver tissue :

    • increases transcription of PEPCK

      • stimulates glyceroneogenesis

  

   

• Thiazolidinediones :

  • reduces the levels of fatty acid in the blood and increase sensitivity to insulin

  • activate PPAR𝛾

    • induces the activity of PEP carboxykinase

  • increase the rate of glyceroneogenesis

    • thus increasing the resynthesis of triacylglycerol in adipose tissue

    • and reducing the amount of free fatty acid in the blood

Summary

• Triacylglycerols are formed by reaction of two molecules of fatty acyl–CoA with glycerol 3-phosphate to form phosphatidic acid

  • this product is dephosphorylated to a diacylglycerol

    • then acylated by a third molecule of fatty acyl–CoA to yield a triacylglycerol

• The synthesis and degradation of triacylglycerols are hormonally regulated.

• Mobilization and recycling of triacylglycerol molecules result in a triacylglycerol cycle. Triacylglycerols are resynthesized from free fatty acids and glycerol 3-phosphate even during starvation. The dihydroxyacetone phosphate precursor of glycerol 3-phosphate is derived from pyruvate via glyceroneogenesis.

Two Strategies for Phospholipid Biosynthesis

1. CDP is attached to Diacylglycerol forming CDP-DAG

   • used to make Phosphatidylglycerol , cardiolipin , inositol

2. CDP is attached to the headgroup forming CDP-Headgroup

   • used to make phosphatidylcholine , phosphatidylethanolamine

Kennedy Pathway

PC and PE

• PE :

  • CDP-DAG ➡️ swap cytosine for serine ➡️ burn off a CO2 = Phosphatidylethanolamine

• PC :

  • PE ➡️ attach 3 carbons using 3 adoMet ➡️ Phosphatidylcholine

Interconversion of PS , PC , and PE

• Base exchange :

  • swaps serine for choline

  • swaps serine for ethanolamine

• PS is synthesized via base exchange in eukaryotic cells

• What is the precursor for glycerophospholipids and triacylglycerol ?

  • Phosphatidic Acid !!!

1. How does FFA or DG contribute to impaired insulin signaling response?

   • circulating FFA & adipokine TNFα may ↑ serine phosphorylation of IRS proteins, causing impaired insulin signal transduction.

2. What predisposes individuals with metabolic syndrome to develop type 2 diabetes?

   • obesity , insulin resistance

3. What is the action of mechanism of metformin?

   • Inhibits mitochondrial Complex 1 ➡️ increases AMP ➡️ activates AMPK

     • AMPK ➡️

       • activates lipogenic gene expression ➡️

         • increases fatty acid mobilization and oxidation

         • inhibits synthesis of glucose , FA , and sterols

       • inhibits lipid and cholesterol synthesis

       • increases the translocation of glucose transporter GLUT4 to the cell surface

4. What is metabolic syndrome?

   • cluster of metabolic disorders

     • obesity , high blood pressure , elevated blood sugar levels , high triglyceride levels , low HDL

   • leads to high risk of heart disease , etc

5. How is acetyl-CoA transported out of mitochondria? Explain the shuttle system

   • must be converted to citrate to exit mitochondria. Then in cytosol citrate can be lysed to convert back.

6. In fatty acid synthesis , there are 7 dehydration steps required for palmitate , why only 6 net $H_{2}O$$H_2O$ ?

   • Seven rounds of the 4-step lengthening reactions produces palmitoyl-ACP

   • In the termination Reaction :

     • Palmitoyl-ACP is hydrolyzed by a thioesterase to release a free palmitate

     • This is why it there are only 6 net H2O molecules.

       • one gets "burnt" here to hydrolyzes free palmitate

7. What is the rate-limiting step of fatty acid synthesis ?

   • Acetyl-CoA carboxylase step

8. How is acetyl-CoA carboxylase regulated ?

   • Inhibitor = palmitoyl-CoA

   • Activator = Citrate

9. What is the primary metabolic source of the reducing power required for fatty acid synthesis and desaturation ?

   • NADPH = reducing agent / electron donor

   • Synthesized in pentose phosphate pathway and also from malic enzyme

10. What are the precursors shared by triaclyglycerols and glycerophospholipids ?

    • Phosphatidic Acid

11. What are the mechanisms of action of dexamethasone and thiazolidinediones on triacylglycerol levels?

    • Dexamethasone = corticosteroid

      • in adipose tissue :

        • decreases transcription of PEPCK

          • inhibits glyceroneogenesis

      • in liver tissue :

        • increases transcription of PEPCK

          • stimulates glyceroneogenesis

    • Thiazolidinediones :

      • reduces the levels of fatty acid in the blood and increase sensitivity to insulin

      • activate PPAR𝛾

        • induces the activity of PEP carboxykinase

      • increase the rate of glyceroneogenesis

        • thus increasing the resynthesis of triacylglycerol in adipose tissue

        • and reducing the amount of free fatty acid in the blood

12. What are the strategies to synthesize membrane phospholipids?

    • CDP is attached to Diacylglycerol forming CDP-DAG

      • used to make Phosphatidylglycerol , cardiolipin , inositol

    • CDP is attached to the headgroup forming CDP-Headgroup

      • used to make phosphatidylcholine , phosphatidylethanolamine