I

ATP ADP

Phosphorylase kinase b ATP

Glycogen Phosphorylase b

Glycogen Phosphorylase a

Glycogen,,

Glycogen Phosphorylase a

Glycogen,,

Glycogen^, + Glucose 1-phosphate

Glycogen^, + Glucose 1-phosphate

Fig. 2.4.2 Signal chain for stimulation of glycogen breakdown by adrenaline (or noradrenaline) and glucagon. Adrenaline or glucagon bind to 7-transmembrane domain G-protein-coupled receptors in the cell membrane. These interact with stimulatory G-proteins (G ) that bind GTP, and which in turn interact with and stimulate adeny-lyl cyclase. This forms cyclic adenosine 3',5'-monophosphate (cAMP) from ATP (see Fig. 2.3.1, Box 2.3, for structure). cAMP binds to, and activates, the cAMP-dependent protein kinase, PKA. This in turn phosphorylates and activates phosphorylase kinase (in its inactive, dephosphorylated form known as phosphorylase kinase b; in its active, phosphorylated form known as phosphorylase kinase a). Phosphorylase kinase a in turn phosphorylates and activates glycogen phosphorylase (again, in its inactive, dephosphorylated form known as phosphorylase b; in its active, phosphorylated form known as phosphorylase a). Glycogen phosphorylase hydrolyses the a-1,4 bonds in glycogen (or strictly phosphorylyses them, using inorganic phosphate, P ), forming glucose 1-phosphate. Phosphorylase kinase is a complex of four types of subunit, one of which (the S-subunit) is calmodulin, a widespread regulatory protein that binds Ca2+. This means that a rise in cytoplasmic Ca2+ concentration will, through activation of phosphorylase kinase, activate glycogen breakdown.

metabolism are listed in Table 2.5. The signal chains that lead from binding of insulin to alteration of gene expression are complex and will not be described here although some at least involve GSK3 (see Fig. 2.4.2 in Box 2.4). Others involve increased expression of the sterol-regulatory element binding protein (SREBP), SREBP-1c. (The SREBP system is described further below.) SREBP-1c is involved in regulation of lipogenic genes by insulin (leading to fat synthesis rather than fat oxidation).

These mechanisms are involved in adaptation to reduced or increased food intake, or a change in dietary composition, over a period of one or more days, but may play little role in the (major) changes in metabolic flux that occur after meals or during exercise.

ACTIVATION 1 INHIBITION

Adrenaline Glucagon | Insulin p-adrenergic \ Adenylyl V Glucagon I N. Insulin receptor /. . ._cyclase_. . receptor |_('] j receptor

+ Protein PKA I phosphatases ATP ADP--p,

HSL active I MAG

Inactive > V. lipase lnactlve

P * JL Fatty acid

V V glycerol

Fatty acid Fatty acid

Fig. 2.4.3 Signal chain for control of hormone-sensitive lipase in adipocytes. Hormone-sensitive lipase (HSL) is the enzyme responsible for regulation of breakdown of triacylglycerol (TAG) stored in adipocytes, to deliver fatty acids to the plasma. HSL is activated by phosphorylation by PKA (see Fig. 2.4.2 for description of the early part of this signal chain). In its active state it catalyses the hydrolysis of TAG to diacylglycerol (DAG), and of DAG to monoacylglycerol (MAG), with release of two fatty acids. A constitutively active MAG lipase removes the final fatty acid. HSL is dephosphorylated and inactivated by constitutively active protein phosphatases. Insulin acts through the signal chain shown in Fig. 2.4.1 to phosphorylate and activate a phosphodiesterase that breaks down cAMP, so reducing the cellular cAMP concentration and allowing inactivation of HSL.

For more details on the components of these signal chains, see Box 2.3.

2.4.1.2 Steroid and thyroid hormones

Steroid and thyroid hormones enter cells and bind to intracellular receptors, which then migrate to the nucleus and bind directly to DNA. The hormone-receptor complexes bind to particular sequences ('response elements') in the promoter regions of the genes whose expression they regulate. Therefore these are 'longer-term' rather than rapid effects. Recently it has been recognised that some steroid hormones also have rapid effects, not mediated by alteration of gene transcription.

2.4.2 Nutrients and control of gene expression

In terms of adaptation to changes of diet, it makes sense that some nutrients themselves (or metabolic products of these nutrients) can alter gene expression. Several systems by which this occurs have been identified.

Table 2.5 Some genes involved in energy metabolism whose expression is controlled by insulin.

Increased

Comments

Suppressed

Comments

Glucose metabolism GLUTI, 2, 3, 4

Hexokinase II

Glucokinase (hexokinase IV) Glyceraldehyde-3-phosphate dehydrogenase

Pyruvate kinase BFE*

Glucose entry into the cell and glycolysis (Box 4.2)

Glucose metabolism

Glucose-

6-phosphatase

Fructose-1,6 bisphosphatase Phosphoenolpyruvate carboxykinase

Amino acid metabolism

Aspartate aminotransferase

Carbamoyl phosphate synthetase I§

Gluconeogenesis^ (Box 4.2)

Amino acid catabolism and urea synthesis (Section 4.1.2.3 and Fig. 4.7)

De novo lipogenesis ATP:citrate lyase

Malic enzymef Acetyl-CoA carboxylase

Fatty acid synthase Lipid metabolism Lipoprotein lipase

Export of acetyl-CoA from mitochondrion (Fig. 4.6)

Synthesis of fatty acids from acetyl-CoA (Fig. 4.6)

Acyl-CoA synthase

Gets fatty acids into cells

'Activates' fatty acids Glycerol-3-phosphate Triacylglycerol synthesis acyltransferase

Transcriptional regulation

SREBP-lc

*BFE: the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (see Box 4.2).

fMalic enzyme is strictly malate dehydrogenase (decarboxylating) (converts malate to pyruvate in cytoplasm: see Fig. 4.6).

^GSK-3 is part of the signal chain (see text).

§Carbamoyl phosphate synthetase I is a key enzyme of the urea cycle (Fig. 4.7). The alternative isoform, carbamoyl phosphate synthetase II, is a cytosolic enzyme involved in purine and pyrimidine synthesis.

This list is rather selective: over 100 genes are known to be regulated by insulin. Based on O'Brien & Granner (1991; 1996).

2.4.2.1 Carbohydrate responsive genes

The expression of some genes is increased in response to increases in carbohydrate availability. Some examples are given Table 2.6. Of course, increased carbohydrate availability also leads to higher insulin concentrations (discussed further in Chapter 5), but it has been shown with cellular preparations that the expression of some genes is increased by glucose without the need for additional insulin. It is now accepted that there are independent pathways for regulation of gene expression by insulin and glucose (Fig. 2.6). The molecular mechanism by which glucose regulates gene expression is not yet completely clear. Clearly glucose itself cannot bind to DNA. Recently a protein has been identified, known as the carbohydrate responsive element binding protein (ChREBP, Fig. 2.6) (other names have been used, for instance ChoRF for carbohydrate response factor). ChREBP is regulated in opposite ways by glucose and by cAMP. Phosphorylation of ChREBP by PKA (see Box 2.3 for definition) leads to inactivation. Dephosphorylation, stimulated by glucose availability, leads to activation, DNA binding and increased expression of the genes whose promoter regions contains the carbohydrate responsive element (ChRE). Glucose has to be metabolised in order to bring about these effects, and there are suggestions that the active metabolite is not glucose itself, but perhaps glucose 6-phosphate or fructose 2,6-bisphosphate. In the pancreatic P-cell, expression of the insulin gene is regulated by glucose (discussed later, Section 5.2.2). Here the immediate transcription factor is known as PDX1, and is different from ChREBP or ChRE identified in liver and other tissues. PDX1 is phosphorylated

Table 2.6 Some genes whose expression is increased by glucose (at a cellular level) or by carbohydrate availability.

Gene Comments

Liver isoform of pyruvate Glycolysis kinase

Acetyl-CoA carboxylase Synthesis of fatty acids from cytosolic acetyl-CoA (see later,

Fatty acid synthase S14 (or Spot 14) SREBP-lc

Lipogenesis*

Transcriptional regulation (In the pancreatic P-cell)

Increased by presence of glucose in the intestinal lumen Transcription factor in pancreatic P-cell increasing insulin gene expression

Insulin SGLT-1 PDX1

*S14 is believed to be involved in lipogenesis in liver and adipose tissue.

Note that the expression of several genes is increased by insulin and glucose acting in concert, and the definition of a 'glucose-regulated' gene is not always clear.

Fig. 2.6 Insulin and glucose control expression of lipogenic genes by independent routes. Insulin probably signals via increased SREBP-lc expression, glucose via a carbohydrate responsiveness element binding protein (ChREBP) (or, in the pancreatic (3-cell, by a transcription factor known as PDX1). Glucagon (in the liver) may antagonise the glucose effect via cyclic AMP. There is considerable 'cross-talk' between the pathways: SREBP-lc expression is also increased by glucose; and SREBP-lc induces enzymes of glucose metabolism such as glucokinase. Based loosely on Koo et al. (2001) with additional information from Kawaguchi et al. (2001).

Fig. 2.6 Insulin and glucose control expression of lipogenic genes by independent routes. Insulin probably signals via increased SREBP-lc expression, glucose via a carbohydrate responsiveness element binding protein (ChREBP) (or, in the pancreatic (3-cell, by a transcription factor known as PDX1). Glucagon (in the liver) may antagonise the glucose effect via cyclic AMP. There is considerable 'cross-talk' between the pathways: SREBP-lc expression is also increased by glucose; and SREBP-lc induces enzymes of glucose metabolism such as glucokinase. Based loosely on Koo et al. (2001) with additional information from Kawaguchi et al. (2001).

when glucose levels rise and moves from its location on the nuclear membrane, into the nucleus to interact with DNA. PDX1 expression is also up-regulated by glucose, giving longer-term control.

The sodium-linked glucose transporter SGLT-1 increases in activity in the small intestine when dietary carbohydrate is abundant. The mechanism seems to involve both increased gene expression and increased translation of mRNA into protein. SGLT-1 is induced by glucose within the intestinal lumen but not by a rise in blood glucose concentration.

2.4.2.2 Fatty acids and gene expression

A particular type of nuclear receptor (or transcription factor, since it regulates gene transcription) is activated by fatty acids. It was recognised as early as the 1960s that an apparently diverse group of xenobiotics (pesticides, etc.) cause the proliferation of the small, oxidative organelles called peroxisomes in rat liver. More recently these compounds have been shown to bind to a nuclear receptor, known for obvious reasons as a peroxisome proliferator-activated receptor (PPAR). We now know that the normal, endogenous ligands for PPARs (i.e. the substances that normally bind to and activate these receptors) are fatty acids, or compounds derived from fatty acids such as certain prostaglandins. These PPARs seem to be a way of regulating gene expression according to the availability of fatty acids. There are three major isoforms of PPAR, described in more detail in Table 2.7. The overall effect of increased fatty acid availability is that fatty acid oxidation is up-regulated in the liver through PPARa, while fatty acid storage as triacylglycerol in adipose tissue is also increased through PPARy.

Table 2.7 Peroxisome proliferator-activated receptors (PPARs): tissue distribution and effects of activation.

Receptor

Other names

Main tissue distribution

Genes whose expression is increased by PPAR activation

Genes whose expression is suppressed by PPAR activation

PPAR-a

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Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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