MB ChB Year 1: Nutrition and Energy

Lecture 22: Specialised metabolism of differentiated tissues.

Dr John Illingworth

Click here to download the printed handout sheets for lectures 21 & 22.

Differentiated tissues have specialised biochemical functions. Individual enzyme activities may be markedly raised or lowered in particular organs, or coded by tissue-specific genes with unique regulatory properties.

Although the absolute enzyme activities may differ by two or three orders of magnitude in different tissues, groups of related enzymes often maintain constant proportions to one another. This suggests that they are controlled as a unit, and their genes may share regulatory motifs.

Differentiated tissues cooperate with one another, but this may involve doing opposite things at the same time: e.g. Cori cycle between liver and skeletal muscles, ketone metabolism, amino acid metabolism in starvation.

Cori cycle

Lactate is normally produced only by red blood cells and type 2B muscle fibres, which have very few mitochondria. It is almost all recycled into glucose by the liver, and the overall process is known as the Cori cycle. Type 2B muscle fibres are only recruited during very strenuous exercise, when blood lactate concentrations may rise sharply. Under these conditions some of the surplus lactate may be oxidised by highly aerobic tissues, such as heart muscle.

Ketone bodies

Ketone bodies (acetoacetate, hydroxybutyrate & acetone) are produced in the liver during periods of rapid fat oxidation, when the rate of fat breakdown exceeds the capacity of the Krebs cycle to process the resulting acetyl CoA. Acetone is produced by the non-enzymic decarboxylation of acetoacetate and may sometimes be smelt on the breath in acute diabetes. Hydroxybutyrate is an "honorary" ketone, because it is chemically related to acetoacetate, but it is in fact a secondary alcohol. The production of ketone bodies is strongly linked to vigorous hepatic gluconeogenesis. Ketone bodies cannot be metabolised any further by the liver, but they are a useful source of energy for extra-hepatic tissues (including brain) during starvation. There is a physical problem getting VLDL, chylomicrons and free fatty acids bound to albumin across the blood / brain barrier, hence the cerebral preference for the water-soluble sugars or ketones. If the blood concentration of ketone bodies exceeds the renal threshold (ketonuria) during acute diabetes then the resulting disturbance of salt and water balance may prove rapidly fatal.

Alanine and glutamine

These two amino acids play a major role in metabolism. Both are present in high concentrations in blood plasma, and they are the principal amino acids released from skeletal muscle during starvation as the muscle wastes away. Most of the other amino acids are converted into alanine and glutamine within the muscle fibres. They are particularly good substrates for gluconeogenesis in enterocytes and liver.

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Differentiated tissues deliver coordinated metabolic responses to major life events including: feeding / starving; waking / sleeping; exercise / rest; pregnancy / lactation; infection; trauma; environmental stress. There is more on this in subsequent lectures.

Division of labour between organs, and transport of metabolites via the blood:

Metabolites Fasting state Fed state

From

To

From

To

glucose

liver

brain (mainly)

intestines (from diet)

most tissues

alanine

muscle

liver

glutamine

muscle

intestine

other amino acids

very little available

not applicable

triglycerides

liver (limited supply)

most tissues

intestines, liver

lactate

red cells, type 2B muscle

liver

red cells, type 2B muscle

liver, heart

free fatty acids

adipocytes

most tissues

very little available

not applicable

glycerol

adipocytes

liver

ketone bodies

liver

most tissues

Some key points:

fat fat fat fat blood heart muscle muscle muscle ileum colon spleen stomach liver gall bladder diaphragm brain

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Inter-organ relationships in humans

It is more efficient to concentrate major metabolic activities in a small number of tissues. This arrangement has been selected in all species, including humans, because specialisation permits higher local substrate concentrations and more rapid catalysis. There is a further advantage where most tissues are resting most of the time, because the available cardiac output, and hepatic support services (for example, lactate re-cycling) can be focussed towards "mission critical" activities which receive the lion's share of the available blood supply.

Use the mouse to point at the tissues illustrated on the right, to review details of their metabolic activities under different physiological conditions. First of all, write down what you think will happen, then review your answers from the screen.

 tissue feeding fasting

 hepatocytes

 

 

 skeletal muscle
(types 1, 2A & 2B)

 

 

 cardiac muscle

 

 

 brain

 

 

 red cells

 

 

 intestines

 

 

 adipocytes

 

 


Examples of key sensors with generalised metabolic effects: AMPK, glucose sensors in pancreas and brain, cytokine sensors in the brain.

AMP-activated protein kinase

AMPK stands for AMP-activated protein kinase. It is the cellular fuel gauge.

AMPK is activated by linear 5' AMP and must not be confused with PKA, or protein kinase A, activated by 3'5' cyclic AMP

There are two different kinds of AMP, which is confusing for students because they are both involved in signalling.

The first kind of AMP is ordinary linear 5' AMP which is formed through the myokinase reaction in the mitochondrial intermembrane space.

ADP + ADP ATP + AMP

ATP is actively exported from the mitochondrial matrix by the adenine nucleotide carrier, which is driven by the mitochondrial membrane potential, so ATP:ADP is normally at least 200:1 in the intermembrane space. Myokinase (also known as adenylate kinase) is freely reversible and the equilibrium constant is close to 1.0 so that cytosolic [AMP] is normally very small, but increases with the square of [ADP] if anything goes wrong with the power supply.

 

 [ATP] x [AMP] 

 

Keq = 


 = 1

 

 [ADP] x [ADP] 

 

 

 

 [ADP] x [ADP] 

[AMP] = 


 

[ATP]

This makes 5' AMP the ideal cellular emergency signal, indicating an emerging threat to the ATP supply long before serious trouble has developed. When 5' AMP rises, cells abandon activities that are not essential for survival, and concentrate on restoring ATP production by switching on glycogen breakdown, and glycolysis.

Both glycogen phosphorylase and phosphofructokinase are powerfully and directly activated by 5' AMP. This emergency switch over-rides numerous other factors that regulate these important enzymes.

In addition, 5' AMP binds to the protein kinase AMPK, favouring its activation by a variety of upstream kinases including the tumour suppressor gene LKB1. This is currently the subject of intense research activity, since it appears that LKB1 plays an important role in apoptosis and is required for the action of the oral hypoglycaemic drug metformin. AMPK is also involved in obesity and weight regulation, and is activated by by the adipocyte hormone adiponectin described below.

AMPK controls the overall balance between energy production and energy utilisation in all eukaryotic cells. AMPK activation tilts the balance towards energy production. Among many other enzymes, it phosphorylates and inactivates HMG-CoA reductase and acetyl CoA carboxylase (described below). This shuts down cholesterol synthesis and fat synthesis, and promotes fat oxidation.

The following figure was compiled from two recent research papers [Canto et al (2009) and Rutter & Leclerc (2009)]. It shows the central role of AMPK activation in three central planks of modern type 2 diabetes therapy: diet, exercise and metformin.

This is very much a work in progress. The "take home message" is that AMPK is intimately involved in the most effective treatments for type 2 diabetes, even though the precise mechanisms remain to be elucidated.

The second kind of AMP is 3'5' cyclic AMP, which is formed by adenyl cyclase in the plasmalemma under the control of G proteins.

ATP 3'5' cyclic AMP + pyrophosphate

Cyclic AMP works through protein kinase A (PKA). It is massively involved in many kinds of hormone actions all over the body and is much more than a pure emergency signal. Some of this will be discussed in the next lecture.

Cerebral blood glucose sensors

So far we have discussed glucose sensing in the pancreatic β cells. There are additional sensors in the hypothalamus, and also near the solitary tract in the medulla, which are outside the blood-brain barrier. Some neurons depolarise, while others hyperpolarise in response to changes in circulating glucose. At least some of these nerve cells, which depolarise in response to rising glucose, appear to use the same sensing mechanism as β-cells, in that they rely on glucokinase and ATP-sensitive potassium channels to initiate signalling. The cerebral sensors are important for controlling the autonomic nervous system. One major effect is a massive outpouring of adrenalin if blood glucose concentration falls, but they are also able to directly control hepatic metabolism via autonomic nervous connections.

Cerebral cytokine sensors

Most cells can respond to a range of cytokines, but the hypothalamus is in a unique position, with overall responsibility for food and energy supplies, salt and water balance, temperature regulation, immune system activity and reproduction, and in ultimate command of both the immune system and the autonomic nervous system. Strategic assessments made in the hypothalamus have effects throughout the entire body. The key information is provided by insulin (size of last meal), leptin (size of food reserves), interleukin-1 (immune system activity) plus a host of less important signals that collectively report the overall status of the organism.

Examples of key regulatory enzymes the list is endless and changes with expanding knowledge, but the following are plainly important: glycogen synthase / glycogen phosphorylase; phosphofructokinase / fructose bisphosphatase; pyruvate kinase / PEPCK; pyruvate dehydrogenase; pyruvate carboxylase; glutamate dehydrogenase; acetyl CoA carboxylase; HMG CoA reductase.

These enzymes are often located at natural bottlenecks, and form the interface between major divisions of metabolism. They provide obvious opportunities for control.

Pyruvate dehydrogenase: this important mitochondrial enzyme is the final "committed" step at the end of the glycolytic pathway. Up to this point it is always possible to get back to glucose, but once through PDH there is no return. PDH is regulated by a kinase / phosphatase cycle. PDH is a huge multi-enzyme complex and the reaction mechanism involves about half of the B group vitamins (see Nelson & Cox, page 569) Which B vitamins, exactly?

Oxoglutarate dehydrogenase (OGDH) in the Krebs cycle has an almost identical protein structure and reaction mechanism to PDH, but the regulation of OGDH and PDH are completely different.

Anaplerotic reactions: (see Nelson & Cox, page 584-6) The Krebs cycle on its own neither consumes nor produces intermediates (it is after all a cycle) but Krebs cycle intermediates are formed during amino acid oxidation and are also the starting point for the synthesis of both glucose and fats. It is therfore necessary to have mechanisms for filling and emptying the cycle to cope with metabolic demands. These ancillary reactions are collectively known as anaplerotic reactions.

The most important of these is pyruvate carboxylase, an ATP and biotin-dependent enzyme that fixes carbon dioxide into pyruvate, making oxaloacetate:

Pyruvate carboxylase is an intramitochondrial enzyme that is powerfully activated by acetyl-CoA, and in healthy people ensures that there are always sufficient Krebs cycle intermediates to meet the needs of the organism.

Acetyl CoA carboxylase is another very important CO2 fixing reaction in the cytosol. It has an almost identical mechanism to pyruvate carboxylase and produces malonyl CoA which is required for the biosynthesis of fats. Malonyl CoA also inhibits carnitine palmitoyl transferase 1 (CPT1, see lecture 19) and helps to control the rate of fat oxidation. If this mechanism could be disabled it could help people to lose weight. This area is being actively investigated as a means to control obesity.

Reaction details: or look for on the metabolic summary chart

Acetyl CoA carboxylase is one of the numerous enzymes that is phosphorylated and disabled by AMPK as part of its general strategy to switch off biosynthetic processes and switch on energy-yielding pathways.

The most important processes that empty the Krebs cycle are gluconeogenesis in liver and kidney, and lipogenesis in liver and adipose tissue.

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Levels of metabolic control from short-term to long-term:

  1. allosteric effects on enzyme activity [milliseconds]

  2. covalent modification (e.g. protein kinase A) [seconds]

  3. sub-cellular location (e.g. GLUT4 in response to insulin) [minutes, but see note]

  4. enzyme synthesis (induction and repression) [hours or days]

Note: neurotransmitter release from synaptic vesicles can be very fast, where there is a premium on speed.

*** Disequilibrium is required for metabolic control ***

This seems so obvious that it is superfluous to mention it, except for the numerous misleading claims about regulatory systems that are actually close to equilibrium. It is only possible to regulate reactions that would otherwise proceed spontaneously with a large negative DG. If a process has almost reached equilibrium regulation will make no difference to the outcome. Consider a large hydro-electric scheme: do we put the sluice gates in the concrete dam, or in the middle of the lake, or downstream on the way out to the sea?

Some regulatory enzymes: click here to visit the interactive metabolic flow chart.

You are not expected to learn this table! It is in any case incomplete because new regulatory enzymes are being discovered every day. It is here for reference, and lists some of the important regulatory enzymes governing metabolism in mammalian liver, and the types of control that are observed.

Enzyme name Metabolic pathway Type(s) of regulation Activators & inducers Inhibitors & repressors

1. Pyruvate dehydrogenase

aerobic glycolysis

covalent modification

Ca++

ATP, NADH & acetyl-CoA

2. Pyruvate carboxylase

gluconeogenesis & lipogenesis

allosteric

acetyl-CoA

-

3. Pyruvate kinase

glycolysis

allosteric & covalent mod

fructose -1,6-bisphosphate

alanine, ATP & cyclic AMP

4. PEPCK (PEP carboxykinase)

gluconeogenesis

gene expression

corticosteroids, cyclic AMP

insulin

5. ATP:citrate lyase

lipogenesis

gene expression

insulin

corticosteroids

6. Acetyl-CoA carboxylase

lipogenesis

gene expression, covalent mod. & allosteric

insulin, ??? citrate

corticosteroids, cyclic AMP? palmitoyl CoA

7. Phospho fructokinase

glycolysis

gene expression & allosteric

insulin AMP, F-2,6-bisP

corticosteroids ATP, citrate

8. Fructose-1,6-bisphosphatase

gluconeogenesis

gene expression & allosteric

corticosteroids? ATP, citrate

insulin? AMP, F-2,6-bisP

9. Triglyceride lipase

lipolysis

covalent modification

cyclic AMP

-

10. a-glycero phosphate acyl transferase

lipogenesis

covalent modification

-

cyclic AMP

11. "malic" enzyme

lipogenesis

gene expression

insulin

corticosteroids

12. fatty acid synthetase

lipogenesis

gene expression

insulin

corticosteroids

13. Isocitrate dehydrogenase (NAD-linked)

Krebs cycle

allosteric

ADP, calcium

ATP, NADH. NADPH

14. Oxoglutarate dehydrogenase

Krebs cycle

allosteric

calcium

-

15. Glutamate dehydrogenase

nitrogen excretion

allosteric

ADP, GDP

ATP, GTP

16. Carbamyl phosphate synthetase

nitrogen excretion

gene expression allosteric

N-acetyl glutamate

-

17. Glycogen phosphorylase

glycogen breakdown

covalent mod. allosteric

cyclic AMP, calcium, 5'AMP

-

18. Glycogen synthetase

glycogen synthesis

covalent mod. allosteric

glucose-6-phosphate

cyclic AMP calcium

19. Glucokinase (liver,
pancreatic b-cells)

blood glucose control

gene expression

insulin

cortisol, cyclic AMP

20. Glucose-6-phosphatase
(liver only)

blood glucose control

gene expression

cortisol, cyclic AMP

insulin

21. HMG-CoA reductase

cholesterol synthesis

gene expression

insulin, thyroxin

glucagon, cortisol, cholesterol


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Examples of key DNA binding proteins with pleiotropic effects. Tissue selective gene expression, and the selective responses to cytokines depend on specific DNA binding proteins which regulate gene expression. There may be literally thousands of the these proteins, which are themselves expressed in a tissue selective fashion. We only have time to examine a few representative examples. PPAR-alpha;, PPAR-γ, CREB, NRF1 and PGC1 act in a combinatorial manner and have particular significance for intermediary metabolism You will not find much in text books, but you can find more information very easily by searching OMIM.

PPAR stands for "peroxisome proliferator activated receptor". Peroxisome proliferators are a diverse group of oils and lipids, used for example as drugs, plasticisers and industrial chemicals, which were found to increase the number of peroxisomes in cells. They often have branched chains or other unusual chemical structures and some of them cause cancers. Their common feature is that they are difficult to oxidise using the conventional mitochondrial pathways, and need a preliminary softening up in the peroxisomes.

PPAR-α stimulates transcription of fatty acid oxidation genes in mitochondria, peroxisomes and microsomes. It is the nuclear receptor for 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 sequence termed the c-AMP response element (CRE) switching on transcription of gluconeogenic enzymes and PGC1. See subsequent lectures for CREB and PEPCK.

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

PGC1 is a transcriptional co-activator for PPAR-g 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.

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Liver has a special role in the regulation of blood glucose. It has a volatile glycogen store and it can switch rapidly between glycolysis to gluconeogenesis. Glucose can readily cross the plasmalemma in either direction.

Liver is the a major location for steroid production, and disposal via the bile.

Liver is the major site for amino acid catabolism and urea synthesis.

Liver is the source of VLDL.

VLDL stands for very low density lipoprotein. The free fatty acids needed to make the triglyceride component in VLDL may be synthesised in the liver from carbohydrate precursors, or they may be derived from circulating free fatty acids bound to albumin. As a result, VLDL are produced during both the fed and fasting states. Contrast this with chylomicrons, which are produced by intestinal cells and are only found in the circulation after a fatty meal.

Liver mitochondria are the source of ketone bodies.

Remember that although the liver makes ketone bodies, it cannot use them. They can be activated by many other tissues (including brain) and oxidised via acetyl-CoA to generate ATP.

Adipose tissue provides a blood glucose sink after meals and a long term energy store. It secretes two important cytokines: leptin and adiponectin. Leptin advertises the size of the fat reserves and is detected by the hypothalamus. Adiponectin is anti-inflammatory, activates AMPK and enhances the effects of insulin.

Leptin is a 16-kD secreted protein that acts mainly on the hypothalamus to inhibit food intake and stimulate energy expenditure. Genetic defects in human leptin production are extremely rare, but when present cause severe hereditary obesity. Leptin output normally rises with increasing fat cell mass. Female leptin levels are significantly greater than those in men. Leptin has signifcant effects on the male and female reproductive systems, and may be involved in the menarche and in hypothalamic amenorrhea due to strenuous exercise or low weight. It also has effects on prostate growth. Leptin activates AMPK in muscle tissue, but reduces AMPK activity in the brain. Leptin has a variety of other functions, including the regulation of hematopoiesis, angiogenesis, bone formation, wound healing, and the immune and inflammatory response. The leptin receptor belongs to the cytokine receptor family and has a single-transmembrane-domain. Receptors are widely distributed and occur in alternatively spliced forms, which may mediate different actions.

Adiponectin is a 33-kD secreted protein that stimulates energy metabolism, enhances insulin sensitivity, and reduces body weight. It is structurally related to collagen and forms triple helices and high molecular weight adducts. Adiponectin binds to cell surface receptors which ultimately stimulate AMPK-kinase and thereby phosphorylate and activate AMPK. This suppresses gluconeogenesis and increases glucose and fatty acid catabolism. Adiponectic receptors also increase PPAR-α but the detailed signalling pathways are unknown. Adiponectin can be taken up by the brain, which responds by reducing food intake and increasing whole body energy expenditure. Adiponectin opposes some of the biochemical effects of TNF-α and has anti-inflammatory actions on blood vessel walls. It may have a role in adipocyte differentiation. Adiponectin output normally falls with increasing fat cell mass in otherwise healthy patients. This effect may contribute to the insulin resistance and inflammation associated with obesity, but adiponectin is unexpectedly low in anorexic and bulimic patients.

Adipocytes secrete pro-inflammatory cytokines such as TNF-α which may also help to explain the association between obesity and inflammatory arterial disease.

Striated muscle: distinguish between the fibre types. It is a source of amino acids during starvation. Muscle can partially degrade amino acids to Krebs cycle intermediates, but has no urea cycle, so it must transaminate the end products and tends to export alanine and glutamine to the blood. These two amino acids are particularly good substrates for gluconeogenesis in liver. Muscles need insulin to use glucose at low work loads, but the requirement is relaxed during vigorous exercise.

Muscle glucose uptake during exercise can make a useful contribution to overall glycaemic control in type 2 diabetes. Such patients are strongly encouraged to lose weight through a combination of diet and increased exercise, with drug treatment and insulin only as the last resort.

Cardiac muscle is specialised for long term sustained energy production from a wide range of substrates. Highly aerobic. Mitochondria may occupy 40% of the cell volume. Can oxidise lactate, but needs insulin to use glucose at low work loads.

Skeletal muscle fibre types

Voluntary muscles contain a variety of fibre types which are specialised for particular tasks. Most muscles contain a mixture of fibre types although one type may predominate. The pattern of gene expression within each voluntary muscle cell is governed by the firing pattern of its single motor neurone. Motor neurones branch within their target muscle and thereby control several muscle fibres, called a motor unit. The high precision eye muscles have only a few fibres in each motor unit, but the muscles in your back have thousands. All the cells in a motor unit contract in unison and they all belong to the same fibre type:

Type 1 skeletal muscle or slow oxidative fibres have a slow contraction speed and a low myosin ATPase activity. Continuously active and fatigue resistant. Used for basal activities. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and prefer to use fat as a source of energy.

These cells are specialised for steady, continuous activity. Their motor neurones are often active, with a low firing frequency. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. These are the marathon runner's muscle fibres.

Type 2A skeletal muscle or fast oxidative-glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are progressively recruited when additional effort is required, but are still very resistant to fatigue. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and can use either glucose or fats as a source of energy. These are general purpose muscle fibres which give the edge in athletic performance, but they are more expensive to operate than type 1.

Their motor neurones show bursts of intermittent activity. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange.

Type 2B skeletal muscle or fast glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are only recruited for brief maximal efforts and are easily fatigued. They have few mitochondria and little myoglobin, resulting in a white colour (e.g. chicken breast). They generate ATP by the anaerobic fermentation of glucose to lactic acid. These are sprinter's muscle fibres, no use for sustained performance.

Their motor neurones transmit occasional bursts of very high frequency impulses. These are large cells with a poor surface to volume ratio and their limited capillary supply slows the delivery of oxygen and removal of waste products.

These differences are nicely illustrated by the serial sections from rat diaphragm published by Gauthier and Lowey (1979) J. Cell Biology 81, 10-25. In the figure above, the left hand section was stained for the mitochondrial enzyme succinate dehydrogenase, the centre panel shows direct immunofluorescence against "fast" type myosin, and the right hand section was stained for alkali-stable ATPase activity (i.e. "fast" type myosin). Notice the differences in the fibre diameters, which correlates with their requirements for efficient gas and substrate exchange. Notice also how the mitochondria tend to cluster near the outside of the cells.

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Brain largely dependent on glucose, but can use ketones. It is mechanically difficult to get fats across the blood brain barrier, because the chylomicrons are too big, and free fatty acids are stuck to serum albumin.

Differentiated tissues the basic essentials.

  1. Glucose is a key metabolite, but we only keep limited reserves.

  2. Control of blood glucose is therefore a very difficult balancing act.

  3. Different tissues cooperate and exchange metabolites via the bloodstream.

  4. Contrasting reactions may occur simultaneously in different parts of the body, e.g.

  5. There are TWO kinds of AMP with very different functions:

  6. The hypothalamus continuously monitors blood composition. Metabolic receptors include:

  7. Regulation occurs at bottlenecks where reactions are far from equilibrium.

  8. Pyruvate dehydrogenase is the principal gateway from carbohydrates into fats.

  9. Filling and emptying the Krebs cycle is an important means of control.

  10. Different regulatory mechanisms differ considerably in speed.

  11. A fiendishly complex system of DNA binding proteins regulates gene expression
    (know of CREB and PPARs but do not learn any details).

  12. Hepatocytes:

  13. Adipocytes:

  14. Voluntary muscle cells:

  15. Heart cells:

  16. Brain cells:


Lectures
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
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18
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21
22
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