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BIOC2120 Lectures 2010: Dr. J.A. Illingworth

Integration & Compartmentation of Metabolism

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Lecture 3 topics:

Relationships between hormonal controls, metabolite transport and major metabolic pathways.

Hormonal controls

Adiponectin is a mixture of anti-inflammatory peptide hormones secreted by adipocytes that also regulate energy homeostasis and the metabolism of glucose and lipids. Adiponectin stimulates the phosphorylation and activation of 5'-AMP-activated protein kinase (AMPK) in skeletal muscle and liver. As a result, adiponectin stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, and phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver. By increasing glucose catabolism, adiponectin achieves a reduction of glucose levels in vivo.

Cholecystokinin is a local peptide hormone produced by neuroendocrine cells in the duodenum in response to partially digested food "chyme" released by the stomach. It stimulates gall bladder contraction and digestive enzyme production by the pancreas, and promotes the release of insulin by the pancreatic islets. Cholecystokinin also has important functions within the central nervous system, and is largely responsible for the feelings of fullness and satiation that terminate a meal.

Glucagon is a polypeptide hormone released from pancreatic α cells, and adrenalin is a low molecular weight catecholamine from the adrenal medulla. Both are released when the blood glucose concentration falls. They both act at the target cell plasmalemma via heterotrimeric G proteins, and increase the concentration of 3'5' cyclic AMP (the beta effect). This activates protein kinase A, which phosphorylates a variety of targets in all parts of the cell. In addition, adrenalin, but not glucagon, has a separate alpha effect mediated partly through inositol triphosphate, diacylglycerol and the release of calcium ions from intracellular stores. The ultimate consequences are increased adipocyte lipolysis and hepatic glucose output, maintaining blood glucose and energy levels when food intake is low. Although these hormones have little or no effect on the contractile performance of voluntary skeletal muscle, they greatly increase the strength and frequency of cardiac contractions.

Glucocorticoids are produced by the adrenal cortex, when stimulated by adrenocorticotrophic hormone [ACTH] released by the anterior pituitary gland. They act slowly on the cell nucleus, changing the patterns of gene expression, and ultimately leading to increased protein breakdown, increased lipolysis and increased gluconeogenesis. They have an overall anti-insulin, diabetogenic effect. Corticosteroids have powerful immunosuppressive and anti-inflammatory effects, and are involved in a negative feedback loop with the pro-inflammatory cytokines (see below). They reduce the ability to fight infections but increase the body's tolerance to stress.

Growth hormone is produced by the anterior pituitary, under instructions from the hypothalamus. It is produced mainly during childhood, with reduced amounts during adult life. Most growth hormone is released at night, during sleep, but it is also stimulated by exercise. The major effect of growth hormone is to promote the synthesis of insulin-like growth factors (IGFs) by peripheral tissues, such as liver, skeletal muscle and bone. IGFs increase the rates of amino acid uptake and protein synthesis. They cause cells to grow and multiply. They also have anti-insulin, diabetogenic effects, increasing lipolysis in adipose tissue and glucose output from the liver, and decreasing glucose utilisation by peripheral tissues, except brain.

Insulin is produced by β-cells in the Islets of Langerhans in the pancreas, primarily in response to increased blood glucose, although amino acids and other metabolites are also effective. Insulin release is greatly potentiated by a variety of local gut hormones, caled incretins, and also responds to neural controls. Insulin acts at the plasmalemma of target cells (principally liver, muscle and fat) initiating a tyrosine kinase cascade. This results in the translocation of glucose porters to the plasmalemma, increased glycogen synthesis, and changes in nuclear gene expression. The ultimate effect is a fall in blood glucose and increased deposition of glycogen and fat.

Leptin is another cytokine, produced mainly by adipocytes, that indicates to the hypothalamus that the fat stores are adequate, and that food intake should be reduced. Leptin also stimulates increased energy expenditure, thereby helping to stabilise long term body weight.

Thyroid hormones are a mixture of low molecular weight iodinated tyrosine derivatives (T3 and T4) produced by the thyroid gland under the control of thyroid stimulating hormone (TSH) from the pituitary. Thyroid hormones increase mental alertness and basal metabolic rate. They elevate the number of catecholamine receptors, thereby enhancing catecholamine effects, and stimulate the differentiation and function of brown adipose tissue to generate heat. Thyroid hormone levels do not vary greatly in healthy well-fed humans, but output is greatly depressed in long term starvation. Under these conditions, people adopt a passive, energy conserving lifestyle that may increase the chances of their ultimate survival.

Pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 were originally identified as products of the immune system, but it is now realised that other cell types, such as adipocytes and muscle, are also significant sources. These peptide messengers increase the body temperature and metabolic rate, and mediate the acute response to injury and infection. They may also, under some conditions, promote apoptosis in susceptible cells. In excess, they produce a variety of extremely unpleasant diseases and give rise to the tissue wasting and cachexia (Greek for "poor condition") seen in patients who are seriously ill.

Summary of hormonal effects

  insulin glucagon / adrenalin growth hormone cortisol TNF-α

effects on carbohydrate metabolism

glycolysis

gluconeogenesis

gluconeogenesis

gluconeogenesis

see note

effects on glycogen metabolism

synthesis

breakdown

synthesis

redistribute to muscle

breakdown

effects on protein metabolism

synthesis

breakdown

synthesis

breakdown

breakdown

effects on fat metabolism

synthesis

lipolysis

lipolysis

redistribute to abdomen

lipolysis

Note: the pro-inflammatory cytokines have anti-insulin effects. This is associated with hepatic gluconeogenesis and glycogenolysis, leading to hyperglycaemia. However, cachectic patients are anorexic and most of their blood glucose is rapidly metabolised by extra-hepatic tissues, in contrast to the fasting state where the subjects are extremely hungry and their residual carbohydrate is conserved.

Hormones are rarely present in isolation, and the overall outcomes observed in vivo result from the summation of these partially contradictory effects.

Negative feedback on pituitary hormone release

There are pronounced negative feedback loops controlling the release of the pituitary hormones. The delays inherent in this feedback system lead to a pulsatile pattern of hormone release. There are marked circadian variations in hormone output and blood samples for hormone measurements should always be taken at a consistent time of day.

Growth hormone production is maximal during the early phase of sleep. Output is very high in children but declines in later life.

ACTH and cortisol production is greatest in the early morning, shortly after waking, and is lowest around midnight.

Compare and contrast feeding and starvation

feature glycolysis / lipogenesis lipolysis / gluconeogenesis

principal source

dietary carbohydrates

body fat and muscle

principal product

triglyceride stores

blood glucose and ketones

dominant hormone

insulin

glucagon, adrenalin

3í5í cyclic AMP

low

high

sugar phosphates

high

low

acetyl-CoA, NADH

low

high

mitochondrial export

citrate

malate and / or aspartate

glucokinase

active

 

glucose 6 phosphatase

 

active

glycogen synthase

active

 

glycogen phosphorylase

 

active

phosphofructokinase

active

 

fructose bisphosphatase

 

active

pyruvate kinase

active

 

PEPCK

 

active

pyruvate dehydrogenase

active

 

ATP:citrate lyase

active

 

"malic" enzyme

active

 

acetyl-CoA carboxylase

active

 

fatty acid synthetase

active

 

adipocyte lipase

 

active

muscle proteases

 

active

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The fed state

Here is an extract from the overall metabolic flow chart, showing those processes which are particularly active after feeding, and the key enzymes that are switched off under these conditions. This is a large diagram and you must scroll across to see the full picture.


Notes on fed state metabolism

There will be urea cycle activity under these conditions, but it depends on how much protein you have eaten.

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The fasted state

Here is an extract from the overall metabolic flow chart, showing those processes which are particularly active during fasting, and the key enzymes that are switched off under these conditions. This is a large diagram and you must scroll across to see the full picture.


Notes on fasted state metabolism

Liver and muscle express different genes for pyruvate kinase. The muscle version is active all the time, but the liver enzyme is phosphorylated and INACTIVATED by cAMP dependent protein kinase. This allows glucagon and adrenaline to regulate gluconeogenesis.

Only liver and kidney have glucose 6 phosphatase. Other tissues have glycogen stores but cannot contribute to blood glucose homeostasis. Glycerol kinase is only expressed in liver cells.

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Futile cycles

We will consider three examples - glycogen metabolism, PFK/FBPase & pyruvate kinase all need careful regulation. Living cells normally avoid fully switching on all the enzymes in the complete loop at any one time, but a little bit of cycling gives more precise regulation.




There is competition between pathways for NAD recycled by the respiratory chain. Usually fat oxidation wins out over all the others, and most tissues (except brain, red blood cells and type 2B muscle fibres) prefer free fatty acids over the other substrates. At high rates of fat delivery, liver produces ketone "bodies" (acetoacetate and hydroxybutyrate) from surplus fat. These are water-soluble and can be used by other tissues, including brain.

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Pyruvate dehydrogenase regulation

Pyruvate dehydrogenase (PDH) is the main control point responsible for the marked preference of most tissues for fat. This intra-mitochondrial enzyme exists in active and inactive forms, which are interconvertible by phosphorylation and de-phosphorylation. Fat oxidation produces copious supplies of NADH and acetyl-CoA (which are also produced by the PDH reaction) and the phosphorylation and inactivation by PDH kinase is favoured under these conditions. The enzyme is reactivated by PDH phosphatase which is strongly stimulated by calcium ions. PDH is the final committed step in the glycolytic pathway, because all the preceding reactions can be undone. By default it tends to become inactivated, conserving carbohydrate supplies when free fatty acids are available as substrate. This makes good sense, given the problem of maintaining the cerebral glucose supply during fasting when triglycerides form our major energy store. In an emergency PDH can be switched on again in a few seconds when calcium levels rise.

The detailed regulation of pyruvate dehydrogenase is more subtle than the system shown above. There are at least four isoenzymes of PDH kinase which differ in their tissue distribution and their response to dietary alterations (Peters et al (2001) Am. J. Physiol. 281(6), E1151-E1158) however the overall pattern is that insulin and high carbohydrate diets lead to sustained increases in PDH activity, while high fat diets result in a long-term reduction in pyruvate oxidation.

Other calcium-dependent mitochondrial matrix enzymes include isocitrate dehydrogenase and oxoglutarate dehydrogenase, but these are allosteric effects that do require a protein kinase like PDH.

Urea cycle regulation

The urea cycle is confined to liver cells, where carbamyl phosphate synthase accounts for a significant fraction of the total mitochondrial protein. The regulation of overall nitrogen balance is still not well understood.

The energy-linked transhydrogenase

The energy-linked transhydrogenase is an ubiquitous mitochondrial enzyme that is driven directly by the proton gradient across the inner mitochondrial membrane. Under physiological conditions maintains a high NADPH / NADP ratio, and a low NADH / NAD ratio in the mitochondrial matrix space. It is freely reversible and it is not entirely clear which is the normal direction of flow: it could use the "high pressure" reducing power from NADPH to make extra ATP, or it might use the energy from the transmembrane gradients to displace the NADPH pool to a more reducing state.

NADH + NADP+ + "energy" = NADPH + NAD+

It maintains a much higher NADPH / NADP ratio than the NADH / NAD ratio in the matrix space. [In this respect the situation inside the mitochondria resembles the position in the cytosol, where the NADPH / NADP pool is fed from some very strongly reducing substrates, and is much more highly reduced than the NADH / NAD pool.] Glutamate dehydrogenase has a dual coenzyme specificity while malate and isocitrate dehydrogenases have parallel NADP and NAD dependent forms. This arrangement makes little sense, because it should lead to futile cycling between the NADPH and NAD pools, but it works well and we donít understand why.

Krebs cycle regulation

The Krebs cycle is the central clearing house of intermediary metabolism. Cells have two methods for regulating the Krebs cycle and related pathways:

Filling the Krebs cycle: pyruvate carboxylase. (The enzyme is needed for both gluconeogenesis and lipogenesis and the gene is expressed constuitively in liver, however the enzyme is allosterically activated by acetyl-CoA.)

Filling and emptying: alanine transaminase. (This enzyme is not regulated and operates under near equilibrium conditions. It automatically fills up the Krebs cycle when pyruvate levels rise, and empties it again when pyruvate levels fall. This may be important for citrate regulation of glycolysis and lipogenesis.

pyruvate + glutamate = alanine + oxoglutarate

Emptying the Krebs cycle during lipogenesis: malic enzyme. This enzyme is regulated by insulin at the level of gene expression in liver and adipocytes.

Emptying the Krebs cycle during gluconeogenesis: phosphoenolpyruvate carboxykinase [PEPCK]. This hepatic enzyme is regulated by cAMP at the level of gene expression. [Run a Web of Knowledge search for 'PEPCK AND CREB' and have a quick look at the article by Horike et al (2008) Journal of Biological Chemistry 283(49), 33902-33910 which is available electronically. Follow the links on each page to find the article. Donít get bogged down in the intricate details, but try to get a feel for the way these genes are regulated.]

DNA binding proteins

CREB is an example of a DNA binding protein that regulates gene expression. There may be thousands of the these proteins, and we only have time to examine very few representative examples. One group of nuclear proteins that act in a combinatorial manner have particular significance for intermediary metabolism are PPAR-α, PPAR-γ, CREB, NRF1 and PGC1. This is new work, and you will not find much about these genes in text books, but you can find more information very easily in Annual Reviews, OMIM or Web of Science.

Interaction between these genes is thought to be responsible for the sustained increase in metabolic rate produced by high food intake. A similar effect is also observed in cachexic patients suffering from serious trauma, cancer or life-threatening inflammatory diseases.

PPAR-α stimulates transcription of fatty acid oxidation genes in mitochondria, peroxisomes and microsomes. It is the nuclear receptor for the fibrates, an important class of lipid-lowering drugs that are used to treat hypercholesterolaemia.

PPAR-γ is necessary and sufficient for adipocyte differentiation from fibroblasts, but it also has roles in muscle and macrophages. It is the nuclear receptor for thiazolidinediones, a new class of oral anti-diabetic drugs.

CREB can be phosphorylated by cAMP-dependent protein kinase, after which it binds to an 8-nucleotide palindromic sequence 5'-TGACGTCA-3' termed the c-AMP response element (CRE) switching on transcription of gluconeogenic enzymes and PGC1.

NRF1 switches on transcription of many mitochondrial genes coded in the nuclear genome.

PGC1 is a transcriptional co-activator for PPAR-γ that promotes mitochondrial gene expression in many tissues via NRF1 and NRF2. In particular it stimulates expression of the mitochondrial uncoupling protein UCP1 and genes from the gluconeogenic pathway. PGC1 is a target for pro-inflammatory cytokines such as TNF-α via p38 MAP kinase. In adipocytes PGC1 causes the differentiation of thermogenic brown adipose tissue.

Note the unfortunate use of the same symbol TORC for two entirely different control systems:

symbol

name

alternate

AAs

OMIM

gene map

TORC1

transducer of regulated CREB 1

METC1

634

607536

19p13

TORC2

transducer of regulated CREB 2

CRTC2

693

608972

1q21.3

TORC3

transducer of regulated CREB 3

 

618

608986

15q26.1

mTORC1

mammalian target of rapamycin complex 1

RAPTOR

trimer

607130

1p36.2

mTORC2

mammalian target of rapamycin complex 2

RICTOR

trimer

609022

 

Transducer of regulated CREB 1 (mucoepidermoid carcinoma-translocated 1 or METC1) activates the interleukin 8 (IL8) promoter. IL8 is a pro-inflammatory cytokine: neutrophil activating peptide. METC1 also activates many genes controlled via the cAMP response element binding protein CREB.

Transducer of regulated CREB 2 (CREB regulated transcription coactivator 2 or CRTC2) also activates IL8 and CRE promoters and is claimed to be a key regulator of fasting glucose metabolism. It integrates external hormonal signals from glucagon with internal metabolic feedback mediated by AMPK. See Koo et al (2005) Nature 437, 1109-1114 and the diagram below.

Transducer of regulated CREB 3 is similar to METC1 and CRTC2. All three CREB coactivators share about 30% sequence identity.

Target of rapamycin complex 1 contains TOR (=FRAP, OMIM 601231), LST8 and RAPTOR and controls the overall rate of protein synthesis, cell growth and overall organ size. These are very widely distributed regulators, also present in yeast. The mammalian version is called mTORC1.

Target of rapamycin complex 2 contains TOR, LST8 and RICTOR (AVO3) and controls actin polymerisation. The mammalian version is called mTORC2.

Control of the gluconeogenic program by TORC: in the fed state TORC is phosphorylated and sequestered in the cytosol, but under fasting conditions TORC enters the nucleus and associates with CREB, switching on numerous gluconeogenic genes (such as PEPCK) and also switching on SIK1 transcription. (SIK = salt-inducible kinase, there are at least 3 of these.) SIK1 phosphorylates TORC, terminating glucagon action after about 4 hours. Gluconeogensis places a huge work load on liver cells. If energy supplies are poor, 5íAMP acting through AMPK provides a safety over-ride and switches glucose production off.

Uncoupling in brown adipose tissue:

Most adipocytes are white in colour and contain a single globule of triglyceride surrounded by a thin skin of active cytoplasm. A minority of brown adipocytes have multiple smaller globules of fat and a more extensive cytoplasm that is rich in mitochondria and respiratory pigments, hence their colour. In contrast to white adipose tissue, brown adipose tissue has a very good blood supply. It is very prominent in newborn animals that have high heat losses, and also in cold-adapted and hibernating species. Differentiation of brown adipocytes is promoted by thyroid hormone, which raises basal metabolic rate. Noradrenalin (from sympathetic nerve terminals) stimulates lipolysis in brown adipose tissue as it does in white adipose tissue, releasing free fatty acids. In white adipose tissue the free fatty acids are released into the circulation, but in brown adipocytes they are transported in a cyclical manner across the inner mitochondrial membrane, collapsing the pH and potential gradients and dissipating the metabolic energy as heat. The movement of free fatty acids is catalysed by mitochondrial uncoupling protein UCP1 that has evolved from the adenine nucleotide carrier. For reviews see Jezek (2002) Int. J. Biochem. & Cell Biology 34(10), 1190-1206 and Skulachev (1998) BBA Bioenergetics 1363(2), 100-124.

Mitochondrial uncoupling proteins UCP1 to UCP5 are present in other tissues and have many functions other than thermogenesis. They also help to minimise free radical damage to tissues at high rates of mitochondrial fat oxidation, and they are involved in the regulation of insulin release. See, for example, an excellent paper by Langin (2001) Diabetes, insulin secretion, and the pancreatic beta-cell mitochondrion. New Engl J Med 345(24), 1772-1774.

Don't forget to try the self-assesment tests!

introduction

metabolic map

lecture 1

lecture 2

lecture 3

lecture 4

self-assessment


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