V

Cleavage

Cleavage f ^ Cleavage

Proinsulin nhz

Signal peptide

,nh2

Mature Insulin V 8 ?

Fig. 5.3 Synthesis of insulin. Insulin is first synthesised as one long polypeptide, preproinsulin. The N-terminal portion is a 'signal sequence' that directs preproinsulin into the secretory vesicles. It is then removed (arrows show sites of proteolytic action). Three disulphide bonds are formed between cysteine residues. (These will hold the mature protein in a particular folded structure.) Further proteolytic cleavage releases the connecting peptide, or C-peptide, to produce mature insulin. Insulin and C-peptide are secreted in equimolar amounts from the (3-cell. Some proinsulin is also secreted into the plasma.

ATP-sensitive K channel

Glucose

Insulin

Fig. 5.4 Glucose-stimulation of insulin secretion in the pancreatic P-cell. Glucose enters the cell via the transporter GLUT2 (but see below) and is phosphorylated by glucoki-nase (GK) (hexokinase IV). These steps are similar to glucose utilisation in the liver and allow the P-cell to 'sense' the plasma glucose concentration. Generation of ATP from glucose utilisation closes ATP-sensitive K+ channels in the cell membrane, stopping the outward flow of K+ ions that normally maintains the resting membrane potential (see Box 7.1 for full description of this). This leads to membrane depolarisation and opening of voltage-sensitive Ca2+ channels. Insulin is present in multiple secretory vesicles in the cell, as a crystalline complex in the centre of the vesicle. An inward flux of Ca2+ ions causes exocytosis of the insulin-containing secretory vesicles, and hence insulin secretion. Glucose also stimulates synthesis of new insulin (see Section 2.4.2.1). Although this scenario is true in rodent islets, there is some question over the presence of GLUT2 in human P-cells and it may be that GLUT1 and GLUT3 give the human P-cell sufficient glucose transport capacity (for discussion, see Schuit 1997).

Glucose

Insulin

Fig. 5.4 Glucose-stimulation of insulin secretion in the pancreatic P-cell. Glucose enters the cell via the transporter GLUT2 (but see below) and is phosphorylated by glucoki-nase (GK) (hexokinase IV). These steps are similar to glucose utilisation in the liver and allow the P-cell to 'sense' the plasma glucose concentration. Generation of ATP from glucose utilisation closes ATP-sensitive K+ channels in the cell membrane, stopping the outward flow of K+ ions that normally maintains the resting membrane potential (see Box 7.1 for full description of this). This leads to membrane depolarisation and opening of voltage-sensitive Ca2+ channels. Insulin is present in multiple secretory vesicles in the cell, as a crystalline complex in the centre of the vesicle. An inward flux of Ca2+ ions causes exocytosis of the insulin-containing secretory vesicles, and hence insulin secretion. Glucose also stimulates synthesis of new insulin (see Section 2.4.2.1). Although this scenario is true in rodent islets, there is some question over the presence of GLUT2 in human P-cells and it may be that GLUT1 and GLUT3 give the human P-cell sufficient glucose transport capacity (for discussion, see Schuit 1997).

Insulin, which is synthesised within the cell and stored in secretory granules, is released by exocytosis of these granules - the granule membrane fuses with the cell membrane and its contents are discharged into the extracellular space. The synthesis of new insulin is also stimulated (via the transcription factor PDX1, see Section 2.4.2.1), and if the stimulus (elevated glucose concentration) persists, insulin secretion will be maintained by increased synthesis.

The response of the P-cells to the surrounding glucose concentration may be studied by isolating pancreatic islets and incubating them in medium containing different concentrations of glucose. The characteristic sigmoid dose-response curve for insulin secretion rate against glucose concentration is shown in Fig. 5.5. Insulin secretion is not much increased until the glucose concentration rises above 5 mmol/l, which (by no coincidence) is the normal concentration of glucose in plasma. In other words, an elevation of the concentration of glucose in the plasma above its normal level will result in increased secretion of insulin.

Glucose concentration (mmol/l)

Fig. 5.5 Dose-response curve for the effects of glucose concentration on the secretion of insulin from isolated human islets of Langerhans, studied in vitro. Insulin secretion is stimulated as the glucose concentration rises above about 5 mmol/l (a typical concentration of glucose in the plasma). Based on Harrison, D.E., Christie, M.R. & Gray, D.W.R. (1985) Properties of isolated human islets of Langerhans: insulin secretion, glucose oxidation and protein phosphorylation. Diabetologia 28: 99-103. Copyright Springer-Verlag, with permission.

Glucose concentration (mmol/l)

Fig. 5.5 Dose-response curve for the effects of glucose concentration on the secretion of insulin from isolated human islets of Langerhans, studied in vitro. Insulin secretion is stimulated as the glucose concentration rises above about 5 mmol/l (a typical concentration of glucose in the plasma). Based on Harrison, D.E., Christie, M.R. & Gray, D.W.R. (1985) Properties of isolated human islets of Langerhans: insulin secretion, glucose oxidation and protein phosphorylation. Diabetologia 28: 99-103. Copyright Springer-Verlag, with permission.

Glucose is not the only stimulus to insulin secretion. Insulin secretion is also responsive to most amino acids (to somewhat differing extents), so that after a meal containing protein there is a stimulus for net protein synthesis. Ketone bodies also (somewhat) stimulate insulin secretion that is stimulated by glucose: this could be seen as a mechanism for restraining ketone body concentrations, since increased insulin secretion will inhibit fatty acid release from adipose tissue and ketogenesis in the liver.

There has been considerable interest recently in the effects of fatty acids on insulin secretion. It now seems that fatty acids are essential for normal glucose-stimulated insulin secretion, and that an increase in the fatty acid concentration for a period of one or two hours will potentiate insulin secretion in response to glucose. It is suggested that a fatty acid derivative, perhaps acyl-CoA, is involved in some way in the insulin secretory pathway. However, if elevated fatty acid concentrations are maintained for more than a few hours, the opposite is seen: there is an impairment of insulin secretion. This seems to relate to an accumulation of triacylglycerol within the P-cell. The mechanism is not understood.

Insulin secretion is also modulated by the nervous system, in ways that will be discussed in more detail in Chapter 7.

Insulin circulates free in the bloodstream; it is not bound to a carrier protein. It affects tissues by binding to specific insulin receptors, proteins consisting of four subunits (two a- and two P-chains), embedded in cell membranes (see Boxes 2.3, 2.4). Signal chains linking the binding of insulin to its receptor and an intracellular change in metabolism were covered briefly in Chapter 2.

Insulin is removed from the circulation after binding to the cell surface insulin receptors. These, with their bound insulin, become internalised (i.e.

taken up into the cell) and eventually the insulin is proteolytically degraded. The process of internalisation may have some role in bringing about insulin's actions, but this is not clear. It is also not clear whether some insulin is removed from the bloodstream by processes that do not result directly in metabolic effects. However, what is clear is that about 70% of the insulin reaching the liver is removed in its 'first passage'. This means that the liver is exposed to much higher concentrations of insulin than other tissues or organs. It also means that swings in insulin concentration are to some extent 'damped down' by the time the insulin reaches the general circulation. This emphasises the special relation between endocrine pancreas and liver.

5.2.3 Glucagon

Glucagon is a single polypeptide chain of 29 amino acids. Like insulin, it is syn-thesised initially as a larger protein (a prohormone) called proglucagon. Proteolytic cleavage gives rise to glucagon. (The large proglucagon molecule can be cleaved to release different peptides in the endocrine cells of the small intestine; see Section 5.7). In contrast to insulin, glucagon's major action is to elevate the blood glucose concentration. In fact, it was first discovered as a contaminant of preparations of insulin made from animal pancreases, which caused some batches to have the opposite of the desired blood glucose-lowering effect.

Its secretion from the pancreatic a-cells, like that of insulin from the P-cells, responds to both glucose and amino acids. However, unlike insulin, glucagon secretion is suppressed rather than stimulated by a rise in glucose concentration (although it is stimulated by amino acids). Thus, a rise in the plasma glucose concentration will lead to an increased ratio of insulin to glu-cagon secretion, and a fall in the plasma glucose concentration will lead to an increased ratio of glucagon to insulin. Again, some glucagon is removed on its first passage through the liver, although probably rather less than for insulin (animal experiments suggest around 5-10%). Nevertheless, glucagon probably has no important metabolic effects in any tissue other than the liver.

Glucagon also produces its effects on intracellular metabolic pathways by binding to receptors in the cell membrane. These receptors are coupled, via G-proteins, to adenylyl cyclase, and intracellular effects of glucagon are mediated via cAMP (see Box 2.4).

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