BCH210- Lecture 22 - Gluconeogenesis and diabetes

what effect does low [O2] have on the body and the biochemical

pathways?

-ETC needs O2 -> H2O, pumps H+ for ATP synthase

-so if there is no O2 then no H+ is pumped and therefore no ATP is made

-anaerobic glycolysis -> lactate + NAD+ , but the whole point is so that NAD+ can be made, so glycolysis can continue and we can make those 2 ATP molecules

how does metabolism change under starvation conditions?

-need to make ATP from glycogen, fats and protein, so glycolysis will be on, PDC will be stimulated, beta-ox -> Acetyl CoA -> CAC will occur too it is phosphofructokinase-1 (PFK-1) that will be on

-glycogen synthesis will be off, acetyl CoA carboxylase for fat synthesis will be off (glycolysis works in all cells)

-creatine kinase is a very limited store of phosphates , there will only be a couple seconds of ATP

why is glucose homeostasis important?

-avoid hypo/hyperglycemia, supply cells with glucose (RBC/neurons)

-liver cells generate glucose in GNG**

what can’t be used in gluconeogenesis (GNG)?

-acetyl CoA

anaerobic metabolism

-during periods of limited oxygen, the Electron Transport Chain is inhibited, and anaerobic metabolism occurs

-all energy production in oxidative phosphorylation is inhibited, and the PDC & CAC slow down

-glycolysis is the only means of generating ATP in this condition

-lactate dehydrogenase converts NADH (made in glycolysis) back to NAD+ so that glycolysis (step 6) can continue

what is the biochemical marker/indicator for anaerobic metabolism?

-increased [lactate]

cori cycle

-IN THE BLOODSTREAM: in the RBCs or muscle cell (low O2) (exercising but your not breathing properly): glucose → pyruvate → lactate + NAD+ (to go back into glycolysis)

-then that lactate will travel into the liver (and kidney):

IN THE LIVER: lactate →(LDH)→ pyruvate→ OAA → (CAC)→ glucose and then glucose can be exported to other cells that need it

**side: TAGs → glycerol → glucose in liver as well

-allows for lactate to be used in Gluconeogenesis (GNG), along with other non-carbohydrate precursors

which organelles don’t have a mitochondria?

-RBCs

when does anaerobic metabolism kick in?

-it kicks in when there’s low O2 and a lot of lactate

gluconeogenesis (GNG)

-the liver (90%) and (kidney→ 10%) can make glucose via this pathway

-this pathway is important because your brain can’t use fats for energy, it needs glucose to generate ATP

-anabolic pathway

-needs a lot of energy

-requires NADH

-is an anabolic pathway that uses high energy intermediates and 4 different enzymes (instead of reverse reaction) to bypass the 3 steps in glycolysis that have negative ΔG°´ and are essentially irreversible

-the products from glycolysis will now be substrates for GNG

what are the key substrates in GNG?

-Key substrates include lactate (3C), glycerol (3C) and certain amino acids that can be turned into glucose

-these carbon-containing molecules are readily available in large quantities from the bloodstream

is GNG simply the reverse of glycolysis?

-no, it is not simply the reverse of glycolysis because A ton of energy would be required for the 10 reactions to go in reverse and make glucose (84 kJ/mol)

what are the 3 irreversible steps in GNG

-step 1

what #1- use of pyruvate for GNG irreversible step 1?

step 1. the addition of CO2 from bicarbonate using pyruvate carboxylase, this requires ATP and gives us OAA

step 2. decarboxylation helps add a phosphate, using PEP carboxykinase (which transfers a phosphate over) → take off the CO2 and then add a phosphate onto it, also uses energy and gives us PEP

THESE OCCUR TWICE CAUSE 2 PYRUVATES**

what is the process of fructose-6-phosphate formation?

-2 * PEP goes through the reverse sequence of enzymes in glycolysis to form a DHAP and GAP, which join to form Fructose 1,6-bisphosphate

-Fructose 1,6-bisphosphatase reverses step 3 of glycolysis (PFK-1)

-Fru 6-P is then isomerized to Glc 6-P

**phosphatases always use a water molecule to remove a phsophate from a molecule

what is the process of glucose formation?

-Glucose 6-phosphatase hydrolyzes off the phosphate to produce free glucose that can exit the cell in order to form our free liver

G6-phosphatase

used for the breakdown of glycogen: using Pi -> G1P -> G6P -> (H20)→ Glucose

the liver cell

-can make glucose from:

1. glycogen breakdown

2. gluconeogenesis

-it also releases the glucose for the other cells to use for energy

the muscle cell

-keeps the glucose and uses it for energy

what is the glucose production from 2 pyruvate in GNG

(substrates) 2 Pyruvate (3C) + 4 ATP + 2 GTP + 2 NADH + 6 H2O + 2 H+ —>

(products) Glucose (6C) + 4 ADP + 2 GDP + 2 NAD+ + 6 Pi

what are the high energy molecules used to drive GNG

-ATP, GTP, and NADH

what does gluconeogenesis in the liver use?

-Gluconeogenesis in the liver uses 11 enzymes to make a single glucose

molecule but is overall favourable when energy is available

how does glycerol to glucose production occur?

-glycerol comes in halfway through GNG

-the glycerol (from TAG) that is released during fat mobilization from fat cells can be used and is released in large amounts into the blood

-glycerol has 3 carbons; 2 glycerol molecules can be used to make a

6-carbon glucose entering the pathway as dihydroxyacetone

phosphate (DHAP)

-an anabolic pathway, we doesn’t need much energy

-2 ATPs are needed to drive this anabolic pathway but 2 NADHs are also produced. 2 H2O are still required to remove the phosphates to make 1 glucose molecule

when is GNG active?

-when increased exercising or fasting

what amino acids can be used to produce glucose?

-alanine

-aspartate

-glutamine

what metabolite does alanine produce to make glucose?

alanine → alpha-ketoglutarate (used in CAC) has 5 Cs

what metabolite does aspartate produce to make glucose?

aspartate → OAA (used in CAC) has 4 Cs

what metabolite does glutamine produce to make glucose?

glutamine → pyruvate (product of glycolysis, used for PDC and to make lactate etc.) has 3 Cs

How does the liver know whether glycolysis or GNG is active?

-by the regulation of the liver

how is the liver regulated, so that GNG and glycolysis aren’t active at the same time? (reciprocal regulation)

GLYCOLYSIS: (done through pyruvate kinase, STEP 10)

• high levels of AMP and F-1,6-BP stimulate it

• high levels of ATP, acetyl CoA, and alanine inhibit it

**low energy inhibits anabolic pathways and high energy stimulates them and need ATP/GTP

GNG: 2-step reaction

• step 1: (pyruvate carboxylase) through allosteric regulation

• high levels of acetyl CoA stimulate it

• high levels of ADP inhibit it

• OAA is in between the enzymes

• step 2: (phosphoenol-pyruvate carboxykinase)

• high levels of ADP inhibit it → if we don’t have energy that 2-step reaction is inhibited in GNG

what is the allosteric regulation at PFK-1 and FBP-1 for glycolysis and GNG to be regulated? (reciprocal regulation)

GLYCOLYSIS: (using PFK-1 for step 3)

• high levels of F-2,6-BP and AMP stimukate it

• high levels of ATP inhibit it

-if you need energy AMP can allosterically regulate PFK-1, and alot of energy turns of glycolysis

GNG: (using F 1,6-BPhosphatase)

• high levels of F-2,6-BP and AMP inhibit it (low energy inhibits it)

fructose 2,6-bisphosphate

-the allosteric regulator

-it stimulates PFK-1 in glycolysis

-it inhibits FBP-1 in GNG

-its made by PFK-2/FBPase-2 (adds 2 phosphates to C2)

PFK-2/FBPase-2

-is a tandem enzyme

-controls the formation and hydrolysis of Fructose 2,6-bisphosphate

-it is a single polypeptide with two active sites that can be reciprocally regulated via phosphorylation (has a kinase and phosphatase domain)

-role as a kinase to add the phosphate (covalently modifies the enzyme) OR role as a phosphatase to remove the phosphate

-controls the production and breakdown of Fructose 2,6-bisphosphate for allosteric regulation

what is the reciprocal regulation of glucagon and insulin on glycolysis and GNG?

INSULIN: glycolysis

• high blood sugar stimulates insulin to bind to GPCR, which in all activates protein phosphatase 1 to hydrolyze PFK-2/FBPase-2 complex (dephosporylating it), to turn on the PFK-2 side and inhibit the FBPase-2 side, with PFK-2 active, F6P can make F-2,6-BP activating glycolysis

• gluconeogenesis will not occur in this time

GLUCAGON: GNG

• low blood sugar stimulates it to bind to GPCR, which eventually activates PKA to phosphorylate the PFK-2/FBPase-2 complex, turing off the PFK-2 side and stimulating the FBPase-2 side, which allows F-2,6-BP to be hydrolyzed into F6P, making GNG more active cause F6P can go on to help G6P → glucose → blood

• glycolysis will not be active at this time

what is the goal of reciprocal control in the liver?

-the goal of reciprocal control in liver is to allow either glycolysis or

gluconeogenesis (GNG) to run, not both at the same time

-regulation is achieved by hormone signaling through enzyme

(de)phosphorylation and the availability of energy

type I diabetes

-born with

-lack of insulin production

-no insulin signalling

type II diabetes

-develops over time

-insulin resistance/ insensitivity (cells aren’t responding to insulin)

-receptors not receiving the signal

-a lack of insulin or insulin-insensitivity leads to poor cellular glucose uptake and hyperglycemia. Glucagon signalling can also turn on GNG, worsening hyperglycemia

hypoglycemia

-low blood glucose

hyperglycemia

-high blood glucose

-arises from glucose no being used to make energy or not being stored as glycogen

glycation

-doesn’t need an enzyme happens spontaneously

-other molecules in the body can undergo this (AGEs), resulting in their

modification which may affect their function

hemoglobin A1c (HbA1c)

-is a marker for high blood glucose since RBCs are turned over every 120 days (4 months)

-isn’t modified

what is glucose’s reactivity?

-glucose is highly reactive and can react with the N-terminal Valine residues of the beta chains in hemoglobin

which hormone is stronger, insulin or glucagon?

-insulin is stronger than glucagon

what are 3 reasons why hypoglycemia could arise in someone with Type I Diabetes?

-taking insulin without eating after

-skipping meals in general

-problems with kidney, GNG, glycogen (can’t break it down), liver