Principles of metabolic regulation
7.3 Peptide hormones, neurotransmitters
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Plasma Lactate (mmol/l)
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Plasma 3-OHB (µmol/l)
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Glucose Saline
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Plasma NEFA (mmol/l)
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Time (min) Time (min)
Time (min) Time (min)
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Plasma Glycerol (µmol/l)
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(b) Figure 7.1 (continued)
Hormones have the capacity to affect the activ- ity of these enzymes, and so can ‘turn on’ a cell.
From a hormone action perspective, there are two ways by which hormones influence their target cells:
• Peptide hormones (in effect polypeptides) and the catecholamines are lipophobic and so are unable to pass through the plasma membrane due to the internal domain of the membrane con- sisting of fatty acid chains. Consequently, they affect their target cells by attaching to receptors on the surface of the cell membrane and thereby influencing the cell. Peptide hormones include insulin, glucagon, and growth hormone (GH).
• Steroid hormones arelipophilicand are thereby capable of passing through the target cell
membrane and attaching to a receptor molecule in the cytoplasm, from which protein synthe- sis is stimulated. Examples include cortisol, testosterone, oestrogen, and progesterone.
7.3 Peptide hormones,
include insulin, glucagon, and the hormones produced by the anterior pituitary gland (growth hormone, follicle-stimulating hormone and pro- lactin). Neuropeptides are secreted by some neurons instead of the small-molecule neu- rotransmitters, and include endorphins. The polypeptide growth factors include a wide variety of signalling molecules that control animal cell growth and differentiation.
Peptide hormones, neuropeptides and growth factors, as well as the neurotransmitters adrenaline and noradrenaline, are unable to cross the plasma membrane of their target cells, so act by binding to the surface receptors on their target cell membrane, and thereby influencing the activity of the target cell by means of activating enzymes within the cell.
Figure 7.2 illustrates the general concept of a peptide hormone affecting its target cell. The hormone must first attach to a specific receptor protein on the surface of the membrane. Once that is achieved, the hormone-receptor complex activates an inactive G-protein, situated on the inner surface of the membrane. G-proteins (gua- nine nucleotide-binding proteins) are a family of proteins involved in transmitting chemical signals from outside the cell to result in changes inside the cell. They communicate signals from many hormones, neurotransmitters, and other signaling factors. Signal molecules bind to a domain located outside the cell. An intracellular domain activates a G-protein. The G-protein activates a cascade of further compounds, and finally causes a change downstream in the cell.
G-proteins function as molecular switches.
When they bind guanosine triphosphate (GTP), they are ‘on’, and when they bind guanosine diphosphate (GDP), they are ‘off’. G-proteins regulate metabolic enzymes, ion channels, trans- porters, and other parts of the cell machinery, controlling transcription, motility, contractility, and secretion. Once activated, the G-protein activates an inactive adenylate cyclase, which then converts ATP to cyclic-AMP (cAMP).
Cyclic-AMP is a signalling molecule capable of activating a range of inactive enzymes, one example beingprotein kinase A(PKA).
Hormone Receptor molecule
Plasma membrane
Inactive G-protein Inside
of cell Outside of cell 1.
Inactive adenylate cyclase Receptor binds hormone
Hormone-receptor complex activates G-protein 2.
Active adenylate cyclase cAMP Other reactions Active G-protein
activates adenylate cyclase
3.
Figure 7.2 General schema showing stages of cAMP formation from a hormone
We shall examine three key examples as to how production of cAMP via activation of the G-protein and adenylate cyclase in the follow- ing sections. These include the activation of glycogenolysis through activation of phosphory- lase, of lipolysis through the activation of HSL, and of, glycogen synthesis via the activation of glycogen synthase.
7.3.1 Adrenaline activation of glycogenolysis Adrenaline, a hormone secreted from the adrenal medulla during exercise, as well as being a neu- rotransmitter secreted by neurons, attaches to its receptor on a muscle cell membrane, from where
Adrenaline Receptor Activated G-protein
Adenylate cyclase
cAMP cAMPregulatory
subunit activated pkA
ATP ADP
activated phosphorylase kinase
activated glycogen phosphorylase
ATP ADP
ATP ADP
Glucose-1-P + Glycogen(n-1) Glycogen
P
P Protein
phosphatase
Figure 7.3 Activation of glycogen phosphorylase as in a muscle cell or in a liver cell
it activates a G-protein, which then activates adenylate cyclase, which converts ATP to cAMP in the cytoplasm of the cell. Hence, a molecule on the outside influences processes inside the cell without actual entering the cell (typical of peptide hormone action). Figure 7.3 illustrates this process.
The cAMP then activates an inactive PKA and the active PKA then activates phosphorylase kinase. which in turn activates glycogen phos- phorylase, the enzyme responsible for cleaving a glucose molecule from glycogen. Note that the active phosphorylase kinase and the active glycogen phosphorylase are phosphorylated, i.e.
they contain a phosphate group from an ATP.
It is also worth noting that the active glycogen phosphorylase is inactivated (by dephosphoryla- tion) by the enzyme protein phosphatase.
Regulation of the activities of phosphorylase kinase and protein phosphatase thereby promotes or diminishes the formation of active glycogen phosphorylase. Adrenaline, through its effect on
increasing cAMP, enhances glycogen breakdown, while insulin, through its effect on protein phosphatase, has an antagonistic effect.
7.3.2 Adrenaline activation of lipolysis During exercise, an increase in circulating adrenaline also targets skeletal muscle cells and adipocytes, resulting in the breakdown of TAGs to release fatty acids and glycerol, both of which can be oxidized in muscle and liver.
Figure 7.4 illustrates how this process of lipolysis occurs. Adrenaline attaches to its receptor on the membrane, activating a G-protein, which activates adenylate cyclase, which converts ATP to cAMP, which then activates PKA. The PKA then activates HSL.
The activated HSL is the phosphorylated form, which cleaves a fatty acid from the DAG to form a free fatty acid and a monoacylglycerol (MAG). You should be aware that the DAG has arisen due to the activation of ATGL. This is not shown in Figure 7.4 since the regulation of ATGL has not yet been clearly established, and we wished to focus on activation of HSL.
The final stage of breakdown to release the third fatty acid and the glycerol involves a monoacylglycerol lipase (MGL). It is also worth noting that insulin is a potent anti-lipolytic hormone, which inhibits HSL activation by stimulating the activity of phosphodiesterase (Figure 7.7). Phosphodiesterase converts cAMP to the inactive form, AMP, resulting in lack of PKA and activated HSL.
So far we have seen how hormones such as adrenaline and insulin affect a muscle or a fat cell by activating or inactivating key enzymes (in these instances through phosphorylation and dephos- phorylation). We shall now explore the effect of a hormone, insulin, on glycogen synthesis, and note that the key enzyme in this case is activated by becoming dephosphorylated.
7.3.3 Insulin activation of glycogen synthase During recovery from exercise, and after con- suming a meal containing carbohydrate, insulin
Plasma Fatty Acids &
Glycerol
Cell Membrane
cAMP
TAG DAG FA MAG FA
FA
Glycerol
HSL−
HSL
+ +
+ ATP
Protein Kinase A
HSL = Hormone Sensitive Lipase Cytosol
Adrenaline
Gs β
adenylate cyclase
P
P
Figure 7.4 Adrenaline activation of HSL
concentrations become elevated in response to an increase in blood glucose levels. As with adrenaline, insulin attaches to a specific receptor on its target cells and thereby modifies activ- ities within the cell. However, the situation is different to what we have seen for adrenaline.
The membrane-bound receptor for insulin is a protein consisting of four subunits. Two subunits protrude out from surface of the cell and bind insulin, and two subunits span the membrane and protrude into the cytoplasm (Figure 7.5). These receptors range from fewer than 100 in most cells in our body to more than 1,00,000 in some liver cells.
The binding of insulin to the outer subunits of the receptor causes a conformational change in the membrane-spanning subunit, which is also an enzyme (a tyrosine kinase). The activated subunits add phosphate groups to the cytoplasmic domain of the receptor, as well as a variety of insulin receptor substrates (IRS). The activated IR then phosphorylates IRS-1 and other substrates. IRS-1 then serves as a docking protein for PI 3-kinase, which is activated by this interaction. The cas- cade initiated by PI 3-kinase involves activation of PI 3-K-dependent kinases (PDK) and thenAkt.
The signalling molecule Akt (also known asPKB) brings about a number of insulin-mediated actions, i.e. activation ofglycogen synthase, translocation ofGLUT4from vesicles to the membrane for glu- cose transport, and stimulation of protein synthesis (Figure 7.5).
Once insulin has attached to its receptor and brought about an increase in Akt within the cell, there is an increase in the activity of the enzyme glycogen synthase. This is achieved by dephosphorylation of the enzyme, which is in contrast to the phosphorylation exhibited by phosphorylase (Figure 7.6). The enzyme glycogen synthase kinase-3 (GSK3) regulates glycogen synthase activity by phosphorylating it and so causing inactivation, and in turn is regulated by Akt i.e. Akt inactivates GSK3 and hence the inhibition of glycogen synthase is removed, so enabling it to increase glycogen storage. In addition, Akt stimulates translocation of the glucose transporter, GLUT4, to move from vesicles within the cytoplasm to the membrane and hence promote glucose uptake into the cell.
Consequently, the increase in glucose and the activation of glycogen synthase leads to greater glycogen synthesis.
Plasma Membrane GLUT4
PDK IRS-1
Akt GSK3
mTOR
−ve
−ve
Insulin Responsive GLUT4 vesicle
Insulin Receptor I
Glucose PI 3-Kinase
Glycogen synthase-P (inactive)
Glycogen synthase (active)
Glycogen Stores Translation of mRNA
Protein Synthesis
Figure 7.5 Insulin action showing activation of glycogenesis. Note how insulin activates Akt, which in turn inactivates GSK3 (an inhibitory enzyme for activation of glycogen synthase). In other words, glycogen synthase becomes activated
7.3.4 Insulin inhibition of lipolysis
Insulin is an anabolic hormone, i.e. it promotes synthesis rather than degradation. To this end, insulin is potently anti-lipolytic and favours stor- age of lipids rather than breakdown. It achieves this through activation of Akt, which then acti- vates phosphodiesterase (PDE-3), an enzyme which converts cAMP to AMP (Figure 7.7). If cAMP levels are reduced in adipose tissue, the consequences are that HSL is not activated and hence lipolysis does not take place. This is just what insulin does. So, if an athlete wishes to
‘fat burn’, they should attempt to keep insulin concentrations low, or else they may be wasting their time! This means not ingesting carbohydrates before or during an exercise session.
7.3.5 Insulin stimulation of protein synthesis The stimulation of protein synthesis is a classic, though maybe to some surprising, action of insulin.
Remember that insulin is an anabolic hormone, and so it should not be a surprise that insulin, as well as promoting fat storage (lipogenesis) and carbohydrate storage (glycogenesis), should also promote protein storage (protein synthesis). Loss of the stimulatory effect of insulin on protein syn- thesis contributes to the cessation of growth and to weight loss.
The effect of insulin on protein metabolism is complex and involves changes in both synthesis and degradation. In some cell types an increase in rate of protein synthesis may be detected within minutes of insulin treatment. This response to
Insulin
Adrenaline Activated
insulin receptor
Activated Adenylate cyclase
cAMP ATP Activated PI3-kinase
Activated protein kinase B or Akt
Inactivated GSK-3
−ve
Activated PKA
More glycogen synthase a
INCREASED GLYCOGENESIS
Phosphorylated IRS1
Figure 7.6 Effect of insulin (and adrenaline) on glycogen synthase
insulin occurs within a timeframe comparable to that of other acute actions of the hormone, such as the activation of glucose transport and glycogen synthase activation. The rapid effects of insulin on protein synthesis involve increases in mRNA translation, the process through which the genetic code transcribed in the mRNA template is translated into protein. Translation takes place on ribosomes in a complex series of reactions that can be segregated into three phases – initiation, elongation and termination (review section 4.5.5).
Although the effects of insulin on mRNA trans- lation have received less attention than those on carbohydrate and lipid metabolism, recent studies have increased our understanding of the mecha- nisms involved in the control of protein synthesis.
The activities of several translation factors have
been found to be controlled by insulin, explaining at least in part the stimulatory effect of insulin on translation. A newly discovered signaling system based on the Ser/Thr protein kinase, mammalian target of rapamycin (mTOR), has been found to have a key role in the control of mRNA translation. Protein synthesis and many of the same signaling elements utilized by insulin to control glucose metabolism have been found to be involved in the control of protein synthesis.
With these discoveries has come an appreciation that signaling molecules identified in studies of protein synthesis may turn out to be important in the control of carbohydrate and lipid metabolism.
Figure 7.5 briefly highlights the fact that insulin signaling affects mTOR and results in increased mRNA translation and hence enhanced protein synthesis. It is beyond the scope of this text to go into detail on the insulin and mTOR signaling pathways, so if you are interested you may wish to consult Saltiel and Pessin (2007). Needless to say however, that after a bout of exercise it is considered advisable for athletes to ingest some form of carbohydrate in order to stimulate insulin secretion and thereby promote protein synthesis. The addition of some form of protein too is necessary in order to provide the amino acids required for building up protein. Indeed, there is a case for the addition of branched chain amino acids (in particular leucine) to stimulate the recovery process.