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Fig. 2.1 Different stages at which the amount of a protein present in a cell may be controlled (underlined). For many proteins the major control is at the level of transcription (mRNA synthesis from the DNA template). Not shown in this figure is a further layer of complexity added by alternative splicing of the mRNA after transcription. The initial, nuclear full-length mRNA molecule is a faithful reproduction of all the DNA sequence of the gene. This includes introns (junk DNA) and exons (DNA that codes for mRNA that will be exported from the nucleus). The introns are removed in the nucleus and the ends of the remaining mRNA 'spliced' together before export from the nucleus. The resulting mRNA may start with alternative exons, giving a range of possible transcripts from one gene.

Fig. 2.1 Different stages at which the amount of a protein present in a cell may be controlled (underlined). For many proteins the major control is at the level of transcription (mRNA synthesis from the DNA template). Not shown in this figure is a further layer of complexity added by alternative splicing of the mRNA after transcription. The initial, nuclear full-length mRNA molecule is a faithful reproduction of all the DNA sequence of the gene. This includes introns (junk DNA) and exons (DNA that codes for mRNA that will be exported from the nucleus). The introns are removed in the nucleus and the ends of the remaining mRNA 'spliced' together before export from the nucleus. The resulting mRNA may start with alternative exons, giving a range of possible transcripts from one gene.

Nutrient X "

I effects on_£ene expression Hor01?-n--'-""

New enz 1

Hormone Pr°duct inhihu-

Transporter * Enz 2B E©3

Substrate > F

cycling

Fig. 2.2 Different methods for achieving changes in metabolic flux within a cell.

A hypothetical metabolic pathway is shown. Enz 1, Enz 2, etc. are the enzymes converting substrate A to substrate B, B to C, etc. For many pathways, important control points are often the first step, and also the first step after a branch point (marked *).

Glycogen Glucose Triacylglycerol Ketone bodies

Acetate

Acetate

Acetyl-CoA

/ \ \ Acetoacetyl-CoA Citrate

Fatty acids Acetoacetyl-CoA

Acetyl-CoA

/ \ \ Acetoacetyl-CoA Citrate

Malonyl-CoA

Fatty acids CO2

Ketone bodies ^testera!

Triacylglycerol

Fig. 2.3 A metabolic regulation puzzle. Illustrated are some of the pathways by which acetyl-CoA may be generated, and some of the pathways by which it is utilised. Think about this: what determines its fate in any particular cell at any particular time? And what prevents 'futile' metabolic cycling: e.g. fatty acids make acetyl-CoA, acetyl-CoA makes fatty acids; or ketone bodies make acetyl-CoA, acetyl-CoA makes ketone bodies? How can the control of ketone body synthesis (a pathway active in 'catabolic' conditions) be achieved when the initial steps in the pathway seem similar to those for cholesterol synthesis (active in 'anabolic' conditions)? The answers will be touched upon in passing and by the end of the book all should be clear.

will return to this puzzle in later chapters (see Section 6.4.1.1 and other sections in Chapters 4 and 6).

2.2 Metabolic regulation brought about by the characteristics of tissues

The characteristics of individual cells or tissues 'set the scene' for metabolic regulation. For instance, the metabolic characteristics of the liver mean that it will inevitably be able to take up excess glucose from plasma, whereas other tissues cannot adjust their rates of utilisation so readily. Therefore the liver is likely to play an important role in glucose metabolism whenever plasma glucose levels are high. The brain, in contrast, has a pathway for utilising glucose at a rate that is relatively constant whatever the plasma glucose concentration, a very reasonable adaptation, since we would not want to be super-intelligent only after eating carbohydrate, and intellectually challenged between meals.

Individual tissues and organs will be discussed in more detail in the next chapter, but here we will look at some general features, especially the means by which nutrients cross cell membranes, which differ from tissue to tissue.

2.2.1 Movement of substances across membranes

Cell membranes, as we saw in Chapter 1, are composed of bilayers of phospholipid molecules (Fig. 1.5). Molecules crossing this membrane must pass through both the hydrophilic, polar faces and the hydrophobic interior. In general, this presents less of a problem for non-polar hydrophobic molecules, and many hydrophobic drugs are able to enter cells readily. It has long been assumed that all hydrophobic molecules can enter cells by simple diffusion, but this is now increasingly under question as more and more specific transporter proteins are described. One example which will be common in this book is that of long-chain fatty acids entering cells: this process was for many years thought to occur by diffusion, but within the past few years specific fatty-acid transporters have been described. This should not surprise us, because the movement of molecules in and out of cells by simple diffusion is, by its very nature, not a process over which any control can be exerted. On the other hand, if the movement occurs by a carrier-mediated process, then immediately there are possibilities for regulation: some cells may have more carriers than others, or the number or activity of the carriers may be altered by hormones. General characteristics of the movement of substances across membranes are discussed in Box 2.1.

Box 2.1 Movement of molecules across membranes

The cell membrane, and membranes within cells, are formed from a phospholipid bilayer (see Fig. 1.5). Most biological molecules, especially polar molecules and ions, will not diffuse freely across such a membrane. Instead, there are specific proteins embedded in the membranes, which 'transport' molecules and ions from one side to the other. This box describes some general properties of the movement of molecules across membranes.

A substance will cross a membrane to move from one solution to another if (1) the membrane is permeable to the substance, and (2) there is a concentration gradient in the appropriate direction: i.e. a substance will move from a region of high concentration to a region of lower concentration. (In reality, there will be movement in both directions because of random molecular movements, but the net movement will be down the concentration gradient.)

There are two major means by which such movement may occur: free diffusion, i.e. unassisted movement by diffusion, brought about simply by the overall effect of random molecular motions, and facilitated diffusion (carrier-mediated diffusion), i.e. movement assisted by a specific transporter protein. In addition, there is a third means of movement: active transport, in which substances may move up a concentration gradient - i.e. from a lower concentration to a higher one. This can only be brought about by the supply of energy, either electrical (charge on the membrane) or chemical; for instance, the Na+-K+-ATPase is a membrane protein that hydrolyses ATP and pumps sodium ions out of cells against a strong concentration gradient, and potassium ions in - also against a strong concentration gradient.

The two forms of movement down concentration gradients - free diffusion and facilitated diffusion - can be distinguished by their kinetic characteristics.

Since the movement of substances by a transporter protein is similar in many ways to enzyme catalysis, it has similar characteristics: there is a characteristic affinity of the transporter protein for the molecule, and a maximum rate of transfer of the molecule which will depend, in turn, on the intrinsic 'rate of action' of the protein, and the number of transporters available in the membrane. Thus, if we measure the rate of transport at differing concentrations of the substrate to be transported, we will find a hyperbolic curve similar to a Michae-lis-Menten plot of enzyme action. On the other hand, if transport occurs by free diffusion, there is no limitation to the rate, and it will be simply proportional to the concentration gradient of the substrate across the membrane. These are illustrated in Fig. 2.2.1.

The presence of active transport will usually be identified by its need for energy. Blocking of ATP synthesis (for instance, by inhibition of oxidative phosphorylation) will usually reduce or abolish such transport.

2.2.1.1 Glucose transport

A particularly important mechanism from the point of view of energy metabolism is the way that glucose crosses cell membranes. There are two families of glucose transporters, each family comprising a number of homologous proteins synthesised from different genes and having different kinetic properties. These are summarised in Box 2.2. The 'GLUT' family are all facilitative (passive) transporters, whereas the sodium-glucose cotransporter (SGLT) family are active glucose transporters. SGLT-1, expressed in the small intestine, needs to be able to move glucose from a low to high concentration, in order to ensure virtually complete absorption from the intestinal lumen. This is achieved by

Box 2.2 Transport of glucose across cell membranes

It has long been known that glucose enters cells by carrier-mediated diffusion (facilitated diffusion) rather than by free diffusion across the cell membrane (see Box 2.1 for definitions). In recent years the genes for the specific glucose transporter molecules have been cloned and sequenced; the transporters have been expressed in cell lines and their characteristics studied. There are two families of glucose transporters. The more widespread family consists of passive transporters, allowing the movement of glucose across cell membranes only down a concentration gradient. They are called GLUTn, where n is a number distinguishing different members. There are 12 known members of this family but only five (shown in the table below) have well-characterised function. The other family consists of active transporters in which glucose may move up a concentration gradient (i.e. it may be concentrated by the transporter) because sodium ions, co-transported with the glucose, are moving down a concentration gradient (see Fig. 2.4). These are known as the sodium-glucose cotransporter family, SGLT-n. The expression of all these transporters is tissue-specific, and their properties are an integral part of the regulation of glucose metabolism in the particular tissue.

The effect which the characteristics of the glucose transporter may have on the rate of glucose entry into cells is illustrated in Fig. 2.2.1. (This refers to facilitated diffusion, i.e. passive transport, only.)

With a glucose transporter whose Km for glucose entry is 1.6 mmol/l (e.g. GLUT3), the rate of glucose uptake is relatively independent of the extracellular glucose concentration over the normal, physiological range of plasma

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