Metabolism Lecture 17/18

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short term extracellular regulation by hormones and other factors

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(seconds-minutes)
due to changes in activity of pre-formed enzyme/protein

no change in protein content

change in SPECIFIC ACTIVITY (activity per mol of polypeptide)

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Long-term extracellular regulation by hormones and other factors

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(hours-days)

accomplished by CHANGES IN PROTEIN CONTENT per cell (and sometimes by an ongoing change in specific activity)

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coordinate regulation is often seen between pathways that...

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use or generate common intracellular metabolites

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reciprocal regulation is often seen between what kind of pathways?

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competing pathways:

e.g.
gylcogenolysis/glycogen synthesis,
FA synthesis/FA oxidation, gluconeogenesis/glycolysis

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extracellular regulation is directed by:

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1. METABOLIC SUBSTRATES in the EC milieu (e.g. glucose, lactate, fatty acids)

2. HORMONES in the EC milieu (whose secretion rates in turn may be determined by a metabolic substrate)

3. Coordinate action of both hormone and substrates (coordination ensures that action(s) of hormones are appropriate to availability/non-availability of substrate)

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functional elements required for a cellular response to a particular hormone/substrate

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-hormone receptor,
-"post-receptor" signalling elements
- regulated protein

*[hormone/metabolite] normal and in bioactive conformation

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mechanisms of "short-term" change in protein fxn by hormones and other factors:

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change in [allosteric effector]

change in covalent modification of protein (e.g. phosphorylation)

change in intracellular localization

change in protein: protein association

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mechanisms of "long-term" change in protein fxn by hormones and other factors:

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change in rate of gene transcription

change in rate of mRNA turnover

change in rate of mRNA translation

change in rate of protein degradation

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protein phosphorylation can result in the following changes in function:

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-enzyme specific activity
-protein binding
-binding of proteins to DNA
-ability of Transcription factors to regulate gene expression
-intracellular localization of proteins

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protein kinases

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catalyze the attachment of a phospate (phosphorylation) of a protein

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donor of phosphate group in protein phosphorylation

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usually gamma-phosphate group of ATP (sometimes GTP)

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protein phosphorylation reactions are

reversible or irreversible

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IRREVERSIBle

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Phosphoprotein phosphatases do what?

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DEPHOSPHORYLATION (remove phosphate), liberating protein and inorganic Pi

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types of protein kinases (what aa residues do they phorphorylate)

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(> 500 kinases encoded in human genome)

some specific for serine/threonine

other only catalyze phosphorylation of tyrosite

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types of phosphoprotein phosphotases

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some specific for phosphoserine/phosphothreonine

and others for phosphotyrosine

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"multi-site" phosphorylation

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describes the presence of different "sites" of phosphorylation on a particular protein, that are catalyzed by different kinsases/phosphatases

explains complicated regulation of protein function

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amplification potential of protein kinase

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1. Each kinase (or phosphatase) can catalyze the phosphorylation (dephosphorylation) of many substrate polypeptide molecules

if the phosphorylated (dephophorylated) substrate is an enzyme that is activated by such modification...

each enzyme in turn can generate many substrate products

2. ACTIVATED ENZYME(s) MAY HAVE KINASE/PHOSPHATASE ACTIVITY Many protein kinases/phosphatases are subject to covalent modification by phosphorylation: this created "cascades" of kinase and phosphatase activation/inactivation which also contributes to further amplification

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glucose regulation of glycogen synthesis/degradation

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involves altered enzyme phosphorylation of:
glycogen synthase
and
glycogen phosphorylase
(IN LIVER CELL)

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effect on low blood glucose (or decreased cellular ATP) on AMPK (and its effects)

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low [glucose] --->(+) AMPK

AMPK ---> (+) ATP generation
and ---> (-) ATP utilization

NET EFFECT: (+) cellular ATP

---> (+) ATP generation via:
(+) FA oxidation and (+) cellular glucose uptake

---> (-) ATP utilization
(-) FA and sterol synthesis
(-) cell division (postponed, bc of high energetic cost)

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regulation of de novo FA synthesis by plasma fatty acid involves

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allosteric regulation of:
acetyl-CoA carboxylase
(in liver cell and adipocyte)

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AMPK

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AMP activated protein kinase

acts as metabolic sensor that "reads" availability of ATP and alters rate of ATP generation or utilization appropriately

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AMPK is activated by what?

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when glucose or oxygen is decreased

cellular ATP is decreased (5'-AMP levels rise)

AMPK is activated:
+ATP generation
-ATP utilization

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"counter-regulatory hormones"

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hormones that are antagonized by insulin:
glucagon
alpha-adrenergic catecholamines (norepinephrine)
beta-adrenercic catecholamines (epinephrine)
vasopressin
cortisol
thyroid hormone
growth hormone

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hormones that bind EXTRACELLULARLY to a receptor on the plasma membrane

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insulin
the catecholamines
vasopressin
glucagon

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hormones that bind to INTRACELLULAR receptors

requiring hormone uptake by cell

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cortisol
thyroid hormone

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what are transcription factors?

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intracellular hormone receptors (eg for cortisol and thyroid hormone)

that bind DNA and regulate gene expression

LONG TERM REGULATION

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time frame of regulation initiated by cell-surface receptors

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BOTH short and long term regulation

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types of signaling from cell-surface receptors (OUR FOCUS)

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Receptor Kinases (insulin, growth factors)
G-Protein Coupled Receptors (peptides, neurotransmitters, postaglandins)

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types of events regulated by cell surface receptors

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altered protein phosphorylation:
(then a post-receptor mechanism effects intermediate metabolism)
-cellular trafficking
-enzymes (activated or inhibited)
-protein synthesis
-membrane effects
- DNA/RNA synthesis

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glucagon and epinephrine bind to what kind of receptor?

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G protein-coupled receptors

linked to adenylate cyclase/cAMP

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Components of G-protein coupled receptor

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receptor
+ heterotrimeric G protein
+ effector (eg Adenylate Cyclase)

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extracellular binding of hormone does what to G-protein in GPCR

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leads to displacement of GDP bound tby GTP on G protein

dissociation of the receptor associated G protein into free alpha and beta/gamma subunits

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GTP-bound alpha subunit of G protein

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activates AC, leading to formation of cAMP from ATP

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GDP-bound alpha subunit of G protein

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associates with beta/gamma subunit, inactive

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cAMP (in GCPR pathway)

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2nd messenger

formed by adenylate cyclase (activated effector)

binds to the regulatory subunit of cAMP-dependent protein kinase, liberating its catalytic subunit, which can then phosphorylate key substrates

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OFF singals of GCPR/cAMP pathway

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1. INTRINSIC GTPase of G protein alpha subunit (hydrolysis of GTP to GDP allows reassociation of heterotrimer, terminating signal)

2. Activity of cAMP PHOSPHODIESTERASE
breaks down cAMP to AMP
activation of this enzyme by insulin is one way in which insulin antagonizes the action of hormones that work through this pathway

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"steady state" of intracellular [cAMP} determined by:

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competing activities of adenylate cyclase and cAMP phosphodiesterase

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Phospholipase C is activated by what kind of signal

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a "free" GTP-bound alpha subunit of receptor associated G protein

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Phospholipase C pathway is activated by what hormones?

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alpha adrenergic catecholamines (norepinephrine)

vasopressin

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what does active phospholipase C do?

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cleaves PIP3 into:
IP3 (inositol-3-phosphate) and
DAG (diacylglycerol)

part of PLC pathway,
IP3 leads to liberation of Ca++ from internal stores
DAG, IP3 and Ca++ (act as 2nd messengers and) activate downstream protein kinases

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off signals of PLC pathway

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1. GTPase of G protein alpha subunit (hydrolysis of GTP to GDP which allows reassociation of the heterotrimer, terminating the signal)

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IP3

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inositol-3-phosphate

along with DAG, product of cleavage of PIP3 by activated phospholipase C

induces the release of intracellular Ca2+ stores

along with Ca2+ and DAG activates downstream protein kinases

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DAG

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diacylglycerol

along with IP3, product of cleavage of PIP3 by activated phospholipase C

along with Ca2+ and IP3 activates downstream protein kinases in PLC pathway