Carbohydrates Flashcards
How do control mechanisms differ between organisms?
Basic features are similar; complexity of these varies to match the environment
e. g. bacteria continually adapt due to a short lifetime, humans less so
e. g. carbon cycle, cells working in a paracrine, autocrine or exocrine manner to best cope with environmental demands
What is flux?
Direction of carbon skeletons - determines their fate, e.g. pentose phosphate, ATP production etc
Where are all enzymes for glucose handling?
Within the cell - these regulate fate of glucose and control net direction of movement
Futile cycle?
The energy required to make G6P with no return; this is coordinated to produce useful precursors for energy production further down to recoup these losses
Function of glycolysis and gluconeogenesis
ATP from glycolysis (substrate level phosphorylation) and CAC (mitochondria and some cells, ox phos)
Storage glycogen - muscle and liver
FA and TAG synthesis
Glycolytic cells i.e. those that lack mcs like RBCs, kidney, or that cannot transport FAs e.g. neurones
Other sugar production
What metabolites in glycolysis cross over with other pathways?
G6P
Pyruvate
Acetyl CoA
Glucose transporters
GLUT1-12, specific for tissues/sugar type
GLUT4 = major, in muscle
GLUT2 = liver
Each is regulated differently, but main response is the insulin, especially GLUT4
Structure of GLUTs?
12 transmembrane proteins forming central core to let glucose through
Tissue specific expression through promoter-regulated control; myocyte enhancer factor MEF-2 binds thyroid hormone receptor, dimerises, and binds to promotor of GLUT4
(time-specific control)
Developmental control - GLUT1 switches to GLUT4 from foetal to neonatal (newborn) muscle
Regulation of GLUTs?
Exist in pools in the cells, moved to and from the membrane in vesicles. e.g. GLUT4 in specific vesicles moved to membrane in response to insulin
Endosomal GLUT1/4 vesicles moved to membrane by muscle contraction (calcium signalling) or anoxia (low O2)
Varberg effect?
Anoxia means electron transport chain can’t be used = glycolysis must be used for energy. The switch to this is the Varburg effect
Overall glucose uptake increase due to:
Regulation after food; insulin directs excess to adipose tissue for storage as TAGs
Energy production regulation; exercise/anosmia to fuel movement or replenish glycogen stores
GLUT2
Liver-specific, no regulation of movement to/from plasma membrane
Dependent on glucose conc in blood stream, and is fully reversible
OVERALL REG OF UPTAKE
GLUT tissue-specificity
Compartmentalisation of GLUT within cell
ER partitioning (??)
Subcellular transport e.g. into mitochondria
Concentration gradients in absence of transporters
Glucose - G6P?
Hexokinases phosphorylate to form G6P, G6P-phosphase reverses
G6P is an allosteric inhibitor of HxK, via feedback inhibition
Control of phos/dephosphorylation?
Enzyme presence/absence
Tissue-specific isoenzymes
State - G6Pase increases activty in starved states, hexokinase massively drops
Hexokinase isoenzymes?
I (A), II (B), III (C)
IV (D) = glucokinase, liver, high affinity
Glucokinase?
Saturates at 10-15mM of glucose, greater than physiological glucose concentration so can sense excess/lack
Present in liver, where blood from intestines first arrives
Also in pancreatic B cell, where insulin is released
Regulation of glucokinase?
Glucokinase gene: complex promoter binds many elements for net effect
Transcription: insulin up via SREBP-1c, CREB down via glucagon – PKA
GK protein - compartmentation with GRP
Degradation - stimulated by glucagon
Glucokinase regulatory protein
Glycolysis = in cytosol
GK localises to cytosol in fed states
When not needed, medium-term control occurs through nuclear sequestration by GRP, binding GK
F6P, further down pathway from G6P, activates GRP to sequester GK in feedback inhibition
Insulin and F1P inhibit GRP
Regulation of G6Phosphatase
4 subunits, translocases T1-3 and a catalytic G6Pase unit
T1 = entry of G6P
T3=exit of glucose
T2-exit of P
Complexes hold catalytic unit in an active conformation
G6Pase unit - FKHR and Foxo1a needed for transcription
Foxo1a dephos via insulin signalling = inactivates transcription = glycolysis
F6P - F16BP
Forward = phosphofructokinase PFK, all cells F16BPase = glucogenic tissues e.g. liver, muscle
Phosphofructokinase control
Allosteric control = ATP and citrate inhibit
AMP/Pi and F26BP stimulate
Isoenzymes:
PFK-1 = F6P-F16BP
PFK-2 = F6P-F26BP OR reversal
Role of F26BP?
Stimulates PFK-1
Inibits F16BPase
PFK-2?
Forms F26BP
Bifunctional - single chain with N terminal PFK-2 activity
C terminal F26BPase activity to reverse
Phosphorylation of either end can fold/unfold and expose that activity to bias activity
This can also have isoforms within itself - alternative splicing varies in cells/cell types to change activity ratio
PFK-2 isoforms and regulation?
L-type - PKA phosphorylates, activated by glucagon, inhibiting PFK-2 activity to favour F26BPase
H and i type - AMP-activated PK phosphorylates, by hypoxia and exercise, to favour PFK-2 activity
Long-term, i-type induced transcriptionally by hypoxia
L-type decreased in starvation and diabetes
Phosphoenolpyruvate (PEP) – pyruvate cycle
PEPCK: oxaloacetate — PEP
PK (pyruvate kinase): PEP — pyruvate
PC (pyruvate carboxylase): Pyr — Oxaloacetate
Pyruvate dehydrogenase: Pyr — Acetyl CoA
Pyruvate kinase
Cytosolic, encoded by two genes, two transcripts from each for isoenzymes:
PKL;
R protein, in RBCs
L protein, in liver, kidney
PKM;
M1 protein, in muscle, heart, brain
M2 protein, minorly in most tissues, major in tumours, foetal form too
Regulation of R and L isoenzymes?
Feedforward activation by F16BP
Feedback inhibition by ATP
Inhibited also by AAs, PKA via glucagon, to favour amino acid conversion to glucose in starvation
SREBP-1c linked to L form long-term control via genes
Acetyl CoA (acoa) fates?
Sterols
Ketones
FAs, triglycerides, phospholipids: oxidised in CAC for ATP
Oxaloacetate fates?
Glucose
Transamination for AAs
CAC intermediate replenishing
Pyruvate dehydrogenase (PDH) regulation
Forms acCoA
Regulatory or catalytic
Phosphorylation by PDH kinase = inactive
Dephos by PDH phosphatase = active
PDH kinase and phosphatase regulation
KINASE
By metabolites: activated by acCoA and NADH, products of PDH i.e. feedback inhibition
acCoA also from FA oxidation = cross talk
Inhibited by ADP
PHOSPHATASE
Insulin signalling activates
Also coordinates with FA use in starvation
Glucose-FA cycle
Preferential use of FAs for ATP
FAs = acCoA = CAC = ATP
Citrate from CAC = inhibits PFK to stop F6P transfer
acCoA inhibits PDH, preventing pyruvate – acCoA (direct to other use)
Pyruvate carboxylase (PC)
Required for glucose synthesis in the liver (pyruvate – oxaloacetate precursors)
Mitochondrial localisation and activity - single gene with isoforms produced by tissue-specific alternative promoters
Allosterically activated by acCoA
Pyruvate carboxylase isoenzymes?
Proximal promoter use: liver, adipose tissue for inducible transcription
Increased by glucagon and glucocorticoids
Decreased by insulin
Distal promoter: kidney, constitutive transcription
Phosphoenolpyruvate carboxykinase (PEPCK)
Cytosolic (inducible) and mitochondrial isoforms from two separate genes
Transcriptional regulators: Foxo1, Creb, SREBP, HND etc
SREBP-1c inhibits in fed state
CREB, nuclear receptor upregulate in starvation
PEPCK isoforms and regulation
Cytosolic: conversion of AA to glucose, inducible
Mitochondrial: lactate recycling (Cori cycle), consitutive
Pentose phosphate pathway
Usually less than 10%, 5-50% of glucose catabolism
In cells undergoing:
active cell division e.g. tumours
active FA synthesis e.g. adipocytes
Circular pathway;
G6P – 6 phosphogluconate – ribulose 5 phosphate
Function and regulation of pentose phosphate pathway
Produces:
NADPH - for biosynthetic pathways e.g. FA
Ribose sugars - nucleotides e.g. in dividing cells
Catalysed by two dehydrogenases, G6PDH/6PGDH, regulated by gene transcription of each
Other sugars?
Fructose – F1P by fructokinase in the liver, enters glycolysis
Lactose – galactokinase, for glucose and galactose
Glycogen synthesis
Built on a fixing protein, localisation of which controls place and amount
Addition of UDP-glucose to glycogenin sequentially = glycogen, using UTP and a number of synthases/branching enzymes
Glycogen breakdown
Addition of phosphate to C1 = G1P, isomerised by phosphoglucomutase to G6P
Sequential clipping of sugar residue bonds with inorganic phosphate by glycogen phosphorylase and debranching enzymes
Complexes in glycogen turnover (7)
Glycogenin and other backbone proteins Enzymes for synthesis Enzymes for breakdown Regulatory protein kinases Regulatory protein phosphatases e.g. PP-1G Cytoskeleton interactions Glycogen storage diseases
Glycogen regulation
Storage sites/amounts differ
Most cells = small amounts, short term fuel reserve
Muscle - 200g, longer, in absence of G6P or oxygen
Liver - 70g, in starvation for tissues with absolute requirement e.g. brain
Glycogen synthase regulation
Allosteric regulation in response to external conditions
GSa = active, b = inactive
Protein kinases e.g. PKA (starvation), AMPK (hypoxia), GSK3 (insulin-responsive,blocks PKA and AMPK), Ca-dependent (muscle in anaerobia), inactive to b
Protein phosphatase 1G activates, stimulated by G6P allosterically and through signalling pathways of insulin
Glycogen phosphorylase
GPb = inactive, a = active
PP-1G inactivates, helped by G6P and insulin inactivate
Glycogen phosphorylase kinase phosphorylates b to activate it to GPa, AMP allosterically activates
(OPPOSITE TO SYNTHASE)
Glycogen phosphorylase kinase regulation
GPKa and b again
GPKa - protein kinases activate by phosphorylating, stimulated by cAMP, Ca2+, AMP
=glycogen breakdown
GPKb - protein phosphatase 1G, stimulated by G6P and insulin
SAME AS SYNTHASE
Glycogen compartmentation of regulation
Only one pair of enzymes activated at a time
Phosphorylase activity decreases rapidly with insulin
GS activation delayed until GP inactive
This is latency coordination - may be due to movement of phosphatases in the compartment
CAC regulation
Many metabolic starting points i.e. glucose, FAs, ketones, AAs
Inputs have varying efficiencies e.g. some AAs better, favour FA
Controlled by citrate synthase, ATP and a-ketoglutarate dehydrogenase
Control also spares acCoA and AAs
Carbohydrates to other metabolites?
Pentose phosphate pathway = nucleic acids
F16BP–PEP and acCoA from pyruvate = lipids
Oxaloacetate from pyruvate = AAs = proteins
Isoenzymes in pathway?
GLUT Hexokinases PFK GP glycogen phosphatase GS glycogen synthase PK pyruvate kinase PC pyruvate carboxylase PEPCK
Where does insulin/glucagon regulate?
GLUT Hexokinases GP and GS G6P -- pentose sugars (dehydrogenases G6PDH/6PGDH) PFK PK, PC, PDH, PEPCK
Where does transcription regulate?
Hexokinases
G6P – pentose sugars (dehydrogenases G6PDH/6PGDH)
PFK
PK, PC, PEPCK
Where does SREBP-1c regulate?
Hexokinases
G6P – pentose sugars (dehydrogenases G6PDH/6PGDH)
PDH, PEPCK
Where does phosphorylation by PKA regulate?
Hexokinases
GP, GS
PFK
PK, PC, PEPCK
Where does allosteric regulation occur?
GP, GS
PFK, F16BPase
PK, PDH
Where does compartmentation control regulate?
GLUT
Hexokinase, G6Pase
GS, GP
