MCB 2000 Chapter 17

the glucose problem

• some tissues need glucose every minute of every day, but stores run out fast

brain:

• ~ 120 g/day

• prefers glucose use under normal. about 60% of the body’s resting glucose budget

red blood cells:

• no mitochondria

• obligate glycolysis. no choice but to burn glucose to lactate

liver glycogen

• ~ 100g stored

• enough to cover roughly 12 to 18 hours of fasting. then it’s gone

within a day of fasting, glycogen is gone but glucose demand keeps going. something has to make more

making new glucose

gluconeogenesis: the synthesis of glucose from non-carbohydrate precursors

where: mostly the liver

• over 90% of glucogenesis happens in hepatocytes (especially overnight and short-term fasting). the kidneys do the rest, around 10% (prolonged fasting)

• not in muscle. muscle lack the final bypass enzyme (glucose 6-phosphatase), so it can never release free glucose to the blood. can break down glycogen and use it for itself, but it can’t even export that carbon as free glucose for the brain

when: wherever glycogen can’t keep up

• overnight fasting. sustained exercise. prolonged starvation. stress states

• all situations where blood glucose is falling and the brain still need to be fed

glucogenesis is a liver job. run mostly when food has stopped coming in

three carbon sources

different precursors, different tissues, all converging on the gluconeogenic pathway

• lactate

• alanine

• glycerol

three small carbon 3-molecules, each marking the tissue and condition that produces it

lactate

• C₃H₆O₃

• from working muscle and RBCs

• anaerobic glycolysis produces lactate

• haring working muscle, where O2 supply is outpaced, and red blood cells which have no mitochondria, are the main exporters

• working muscle produces lactate when glycolytic flux outruns mitochondrial oxidation and red blood cells produce lactate continuously because they don’t have any mitochondria

• lactate is sent to the blood then to the liver to be converted back to pyruvate

alanine

• C₃H₇NO₂

• from muscle protein breakdown

• during fasting, muscle proteins are degraded

• the amino acid alanine carries both the carbon skeleton and waste nitrogen from muscle to liver

• muscles transfer nitrogen onto pyruvate, making alanine which travels to the liver

• in the liver, alanine donates its nitrogen for urea synthesis and its carbon skeleton becomes pyruvate for gluconeogenesis

glycerol

• C₃H₈O₃

• from fat tissue (adipose) triacylglycerols

• when fat stores are mobilized, triacylglycerols are cleaved into three fatty acids and one glycerol

• the fatty acids fuel β-oxidation; the glycerol comes here to gluconeogenesis

where each precursor enters the pathway

lactate and alanine enter at the bottom (as pyruvate)

glycerol jumps in halfway up (as DHAP)

Lactate:

• in the liver, lactate dehydrogenase runs in the oxidative direction so in reverse

• this converts lactate back to pyruvate and produces cytosolic NADH

Alanine

• transamination removes the amino group from alanine leaving us w/ pyruvate

• other glucogenic amino acids can enter as oxaloacetate or other TCA intermediates but alanine is the clean pyruvate example

glycerol:

• its phosphorylated and oxidized DHAP which joins the pathway about halfway up

• skips the most expensive lower bypass steps

why not just reverse glycolysis

• three steps in glycolysis release so much energy they cannot be reversed under cellular conditions (extremely exergonic)

  • function as one way steps and pushing them backwards directly would require unrealistic metabolite concentrations

• reversing glycolysis means climbing back over these three cliffs.

• the cell uses different enzymes instead for gluconeogenesis to get around those thermodynamic barriers

• central design principle of the pathway: use the reversible steps of glycolysis but bypass the irreversible ones

the bypass strategy

• four unique gluconeogenic enzymes replace the three glycolytic steps

glycolysis: forwards, downhill

• glucose → G6P (hexokinase/ glucokinase)

• F6P → F-1,6-BP (phosphofructokinase-1)

• PEP → pyruvate (pyruvate kinase)

gluconeogenesis: reverse, with bypasses

• G6P → glucose (glucose-6-phosphatase (bypass 4))

• F-1,6-BP → F6P (fructose-1,6-bisphosphatase (bypass 3))\

  • major cytosolic control point for gluconeogenesis

• pyruvate → OAA → PEP (pyruvate carboxylase + PEPCK (bypass 1+2))

three glycolytic barriers, four gluconeogenic bypass enzymes. the three pyruvate-to-PEP barrier needs two steps to cross

bypass 1: pyruvate carboxylase

• adds a CO2 (supplied as bicarbonate) to pyruvate. mitochondrial matrix. ATP-consuming. biotin-dependent

• converts pyruvate (3C) to oxaloacetate (4C)

• cost: 1 ATP per pyruvate

where: mitochondrial matrix

• pyruvate enters from cytosol via the pyruvate transporter

• the reaction happens inside the matrix

• oxaloacetate is made inside the mitochondria and cannot simply diffuse out

cofactor: biotin

• covalently linked to the enzyme on a flexible arm

• the biotin arms picks up CO2 from bicarbonate (HCO₃⁻) and swings it onto pyruvate

activator: Acetyl-CoA

• allosterically activates by acetyl-CoA

• signal: TCA is saturated

• send pyruvate to glucose synthesis instead

• high acetyl-CoA tells the mitochondria that fuel is abundant and that pyruvate should be diverted towards glucose synthesis rather than further oxidation

step 1: add a carbon. mitochondrial. costs 1 ATP per pyruvate

bypass 2: PEPCK (phosphoenolpyruvate carboxykinase)

• removes a CO2 and adds a phosphate. the rate-limiting, committed step of gluconeogenesis

• converts oxaloacetate to phosphoenolpyruvate (PEP) by removing Co2 and using GTP to install a phosphate

• cost: 1 GTP per oxaloacetate

• pyruvate carboxylase just added CO2, but that add and remove strategy is how the cell crosses the pyruvate kinase barrier

where: cytosol

• once oxaloacetate is in the cytosol (via the shuttle)

• PEPCK takes over here

• oxaloacetate must get here from the matrix back out into the cytosol before PEPCK is going to be able to use it

chemistry: decarboxylation drives it

• releasing CO2 generates a high-energy enol intermediate. GTP donates its phosphate to trap the enol as PEP

• helps dive formation of the high- energy enol phosphate in PEP

regulation

• committed step

• rate-limiting for the whole pathway

• strongly regulated by hormones at the gene-expression level

• when fasting, glucagon promotes PEPCK expression and in the fed state, insulin suppresses it

step 2: remove the carbon you just added, but trap a phosphate in the process. costs 1 GTP per OAA

the oxaloacetate shuttle

• OAA can’t cross the inner mitochondrial membrane. malate can

• OAA hitches a ride as malate. NADH made in the matrix is effectively delivered to the cytosol as a bonus

• the shuttle does two jobs: moves the OAA out of the mitochondria and helps supply cytosolic NADH needed later in gluconeogenesis

  • NADH matters especially when the starting precursor is pyruvate or alanine

  • if the starting precursor is lactate, lactate dehydrogenase already produces NADH directly

why two steps for one bypass?

• PK’s huge ΔG°′ broken into two manageable carboxylation/ decarboxylation steps

• splitting the climb into two smaller, ATP-paid steps is how the cell scales the -31 kJ/mol cliff

• so, the net cost is 2 high energy phosphate bonds per pyruvate, or 4 per glucose just to get from pyruvate back to PEP.

  • why the bottom of gluconeogenesis is the most is the most expensive part of the pathway

bypass 3: F-1,6-BP (fructose-1,6-bisphosphatase)

• hydrolyzes as phosphate off F-1,6-BP. cytosolic. major allosteric regulation point

where: cytosol

• reverse the PFK-1 step of glycolysis (which adds a phosphate to F6P using ATP)

• FBPase-1 does not reverse that by making ATP, instead it hydrolyzes the phosphate from F1,6BP and releases and inorganic phosphate as a product

chemistry: hydrolysis, not transfer

• the phosphate is hydrolyzed off as inorganic phosphate

• this hydrolysis helps to drive the gluconeogenic direction but it also means the energy originally spent by PFK-1 is not recovered

• no ATP is generated; this drives forward vias mass action and ΔG° loss

regulation: F-2,6-BP master switch

• inhibited by fructose 2,6-bisphosphate and by AMP

• activates by citrate

• both enzymes coexist in the cytosol of liver cells — so, potentially futile cycle point

  • if both ran strongly at the same time, the cell would spend ATP adding a phosphate, and then would immediately hydrolyze it off

• F2,6BP activates PFK-1 and inhibits FBPase-1 pushing the pathway decisively in one direction based off of its presence or absence

step 3: a simple hydrolysis. cytosolic. the cells main lever for switching between glycolysis and gluconeogenesis

bypass 4: glucose 6-phosphate

• releases free glucose into the blood. only in liver and kidney. ER lumen, not cytosol

• another hydrolysis reaction

• G6P+ H2O → Glucose + Pi

• no ATP made

where: ER lumen (liver, kidney)

• embedded in the ER membrane with its active site facing the lumen

  • so G6P needs to be transported into the ER, hydrolyzed there and then glucose and phosphate are transported back out

• muscle and brain lack this enzyme — mainly expressed in liver and kidney

  • this is why glycogen is for muscle use only

  • muscle can generate G6P from glycogen but without G6-phosphatase, it can’t be released into the bloodstream

transport: G6P must be imported

• a transporter shuttles cytosolic G6P into the ER

• after hydrolysis, free glucose and Pi are exported back out

consequences: why only liver and kidney release glucose

• without this enzyme, G6P stays trapped in the cell

• muscle uses its own G6P locally; only liver and kidney can resupply blood glucose

step 4: hydrolyze the last phosphate. only liver and kidney can do this, which is why they’re the only blood-glucose suppliers

glycolysis vs gluconeogenesis: where they differ

three barriers in glycolysis. four enzymes in gluconeogenesis. three different compartments

the complete gluconeogenic pathway

from pyruvate at the bottom to glucose at the top, with 4 bypasses across the three compartments

the bill: making one glucose from two pyruvates

• three reactions each happen twice (once per pyruvate)

• each row is per-pyruvate. doubling gives the per-glucose cost

• three reactions x 2 = 6 ATP equivalents

where the cost lands along the pathway

• 2 ATP and 2 GTP at the bottom, 2 ATP plus 2 NADH in the middle. Glucose at the top costs nothing more

• two-thirds of the cost lives in the bottom of third of the pathway: the pyruvate-to-PEP crossing

compared to glycolysis

• glycolysis yields 2 ATP per glucose. gluconeogenesis costs 6 ATP equivalents. A round trip loses 4

• going glucose → pyruvate gives you 2 ATP. coming back costs you 6.

• the 4 ATP differences is the price of irreversibility

precursor matters: glycerol is cheap

• glycerol enters at DHAP, skipping the most expensive part of the pathway

Lactate: enters at pyruvate

• full bill

• pays for PC, PEPCK, PGK reverse, GAPDH reverse

• 3 ATP eq. per lactate

• 6 ATP eq per glucose

alanine: enters at pyruvate (after transamination)

• full bill

• same path as lactate, plus nitrogen of offloaded for urea synthesis

• additional cost, separate budget

glycerol: enters at DHAP

• discounted

• skips PC, PEPCK, PGK reverse

• costs only 1 ATP per glycerol at glycerol kinase

• about 2 ATP per glucose from glycerol

same destination, very different fares. where you enter the pathway determines what you pay

why both pathways cannot run at once

• without reciprocal regulation, the cell would burn ATP in futile cycles

• futile cycle: if both enzymes are active:

  • F6P + ATP → F1,6BP

  • F1,6BP +H2O → F6P +Pi

  • net effect: ATP is hydrolyzed to ADP+ Pi. nothing is built. the cell heats up

the cell solves this by ensuring that when one enzyme is active, the other is shut down

three regulatory pairs

• each irreversible glycolytic enzyme is reciprocally regulated against its gluconeogenic counterpart

pair 2 is the master switch. F2,6BP activates PFK-1 and inhibits FBPase-1 at the same time

spotlight: F2,6BP

• one small molecule, two opposite effects on two enzymes

• when F2,6BP is HIGH: glycolysis runs, gluconeogenesis stops. when it’s LOW: the reverse

hormonal control: the switch you already know

• same PFK-2/FBPase-2 cascade as the glycolysis chapter — both hormones, side by side

glucagon and insulin are opposing signals at every level: receptor, second messenger, phosphorylation state, gene expression

allosteric fine tuning by small molecules

• beyond hormones: the cell’s own metabolite adjust flux in real time

• acetyl-CoA — mitochondrial fuel is high

  • activates pyruvate carboxylase

  • diverts pyruvate to gluconeogenesis

• citrate: TCA cycle is saturated

  • inhibits PFK-1, activates FBPase-1

  • slows glycolysis, speeds gluconeogenesis

• AMP: ATP is being used up

  • activates PFK-1, inhibits FBPase-1

  • speeds glycolysis, slows gluconeogenesis

• ATP: energy charge is full

  • inhibits PFK-1 and pyruvate kinase

  • slows glycolysis when ATP is plentiful

local metabolites tune the pathway in real time . high fuel → make glucose. Low ATP → burn glucose

the integrated picture

• fed and fasted states have completely opposite enzyme activity profiles

• every layer is reciprocal. one state’s ON the other state’s OFF at every level of the control system

the Cori cycle: muscle and liver in partnership

• lactate goes to liver. glucose comes back. the blood is the highway

• muscle exports the problem; liver pays the bill. the cycle keeps muscle running even when it can’t use oxygen fast enough

why doesn’t the cycle violate thermodynamics

• muscle gains 2 ATP; liver pays 6 ATP. the net 4 ATP comes from liver oxidation of fatty acids

muscle: burns 1 glucose, gains 2 ATP

• glucose → pyruvate via glycolysis

• pyruvate reduced to lactate to regenerate NAD+

• net muscle: +2 ATP

• lactate is exported because muscle cannot oxidize it further without enough O2

liver: spends 6 ATP equivalents per glucose rebuilt

• 2 lactate → pyruvate → 1 glucose via gluconeogenesis

• net for liver: -6 ATP equivalents

• those ATP are supplied by mitochondrial oxidation of fatty acids in hepatocytes, which yields plenty of ATP from β-oxidation

whole body: net cost is 4 ATP per cycle

• the cycle is energetically expensive overall (-4 ATP per loop)

• but it lets muscle keep contracting when O2 runs short, and the energy bill is paid from fat stores via the liver

• the cost is acceptable because the alternative is stopping work

muscle borrows energy from liver. liver borrows energy from fat. fat muscle work via the liver as middleman

starvation timeline: how the body adapts

• days 1 to 3: gluconeogenesis carries the load. After day 3: ketone spare glucose and protein

day 1- 3: glycogen depletion, gluconeogenesis dominants

• Hepatic glycogen runs out in t12 to 18 hours. gluconeogenesis ramps up to maintain blood glucose

• precursors: lactate from RBCs and working muscle, glycerol from fat mobilization and amino acid carbon from muscle protein breakdown

• the brain runs on glucose, muscle is being slowly consumed

day 3 onward: ketone bodies enter the picture

• liver starts producing ketone bodies ( acetoacetate, β-hydroxybutyrate) from fatty acid oxidation

• brain shifts gradually ketone as fuel, reducing glucose demand from ~120 g/day to as little as 40 g/day

• gluconeogenesis still runs, but at a lower rate

weeks 1+: protein sparing

• with ketones covering most brain fuel, the rate of muscle protein breakdown drops sharply

• this is the body’s adaptation to extend survival; preserving muscle protects organ functions and lets the person liver for weeks on fat reserve alone

glucose demand drops as ketone bodies take over brain fuel. that’s what lets humans survive prolonged fasts

type 2 diabetes: gluconeogenesis is stuck on

• insulin resistance breaks the off-switch. the liver keeps making glucose even when blood glucose is already high

normal physiology: after a meal

• insulin rises _> suppresses hepatic gluconeogenesis → blood glucose stabilizes

• fasting blood glucose: <100 mg/dL

type 2 diabetes: insulin resistance

• insulin signal weakened gluconeogenesis not suppressed → liver keeps exporting glucose even after eating

• fasting blood glucose: ≥ 126 mg/dL (diabetic range)

T2D fasting hyperglycemia is largely a gluconeogenesis problem. metformin lowers hepatic glucose output