CHAPTER OUTLINE

Circulating glucose derives from three main sources:

• hepatic and some other glycogen stores (glycogenolysis)

• new synthesis from precursors (gluconeogenesis). Gluconeogenesis takes place in the liver (~75–90%) and kidneys (~10–25%) from glucogenic amino acids, especially alanine, and from glycerol, lactate and pyruvate. Principally it involves the synthesis of a 6-carbon compound from various 3-carbon skeletons. Partial oxidative metabolism in tissues like adipocytes and muscle provides lactate and pyruvate but they can also be donated by red blood cells, in which anaerobic metabolism occurs in the absence of the enzymes of the Krebs (tricarboxylic acid) cycle. Triacylglycerol (triglyceride) released from adipocytes during lipolysis provides glycerol.

Regulation of gluconeogenesis can be via changes in the amount of substrate arriving at the liver or by the amount that the liver itself extracts, as well as by regulation within the liver. This process is subject to direct hormonal modulation by insulin, glucagon and catecholamines and indirectly by other hormones.

Hepatic glucose output is ~2.0 mg/kg body weight/min in the resting postabsorptive state, or 200–300 g during the average 24 h period (varying with the availability of glucose from food and with the body’s requirements during exercise). Plasma glucose concentration is determined by the balance between glucose influx into the circulation (principally from food and hepatic glucose production) and peripheral glucose clearance.

Glucose is stored as glycogen, a 70 kg man typically having a total of 700–1000 g (hydrated) glycogen. Most of this is in the skeletal muscle (400–600 g), and liver (60–125 g), with lesser amounts in other tissues. Glycogen is synthesized from both glucose and the gluconeogenic substrates (see above). Glycogen is a polymeric storage form of glucose. Extension of the polymer by the addition of glucose 6-phosphate subunits is catalysed by glycogen synthase, which is the main regulatory enzyme for glycogen synthesis. This enzyme is itself subject to regulation by a dephosphorylation/phosphorylation cycle controlled by a cyclic AMP dependent protein kinase. Glycogen in skeletal muscle can provide fuel for muscle but does not provide a source of glucose for release into the circulation.

Glucose provides approximately 40–60% (on a typical diet in the developed world) of the total fuel expended by the body during a 24 h period. It provides almost all the energy for the central nervous system (especially in well-nourished subjects, although sustained fasting allows cerebral ketone body utilization). During high-intensity exercise and during the 4–6 h postprandial period, glucose is the predominant fuel for the whole body. Glucose is the most efficient fuel for oxidation in terms of the liberation of energy (112.2 kcal or 6 moles ATP per mole of oxygen consumed). Many tissues can use ketone bodies, fatty acids or glucose for their basal metabolism, switching between these different fuels depending upon their availability in the circulation.

Glucose is fully oxidized to carbon dioxide and water in skeletal muscle, the brain and the liver. The brain accounts for most of the glucose oxidized in the fasting state (100–125 g/24 h). In the resting, fasted state, skeletal muscle takes up 10–20% of hepatic glucose output: this is not all oxidized but can be converted to lactate, pyruvate, glycerol or the carbon skeletons of amino acids, much of which subsequently return to the liver and act as precursors for gluconeogenesis. Fatty acids (or their partial oxidation products, ketone bodies) are the prime fuel of resting muscle, heart and liver. Other tissues such as red blood cells, skin, adipose tissue and the renal medulla derive energy from glycolysis to lactate and pyruvate, even in the resting state. Glycolysis to lactate is an anaerobic process to which many cells may resort when faced with hypoxia, for example skeletal muscle during high-intensity exercise. The brain can adapt to the utilization of ketone bodies as a source of fuel during a prolonged fast, but this is far from being an instantaneous process, and the more immediate response depends upon increasing glucose supply as a result of the action of the counter-regulatory hormones glucagon, cortisol, catecholamines and growth hormone. Although each can achieve the objective of restoration of blood glucose supply to the brain, the mechanism for each differs. Adrenaline (epinephrine) and glucagon are the ‘rapid response’ team, activating hepatic glucose formation via glycogen phosphorylation, whilst growth hormone and cortisol are the ‘back up’ team inducing the enzymes responsible for gluconeogenesis. Catecholamines can additionally increase circulating non-esterified fatty acid concentrations by upregulating lipolysis so muscle uptake of glucose falls, leaving more glucose for the brain to use.

In contrast, if the counter-regulatory hormones are present in excess and there is also a relative deficiency of insulin then hyperglycaemia results; infections and trauma are common causes of increased secretion of catecholamines.

The 14 facultative glucose transporters recognized to date are classified into those having high glucose affinity (class I comprising GLUTs 1–4), high fructose affinity (class II, e.g. GLUT5) and novel transporters whose physiology is not yet fully understood (GLUTs 6–14). The different functions of the class I GLUTs are partly predictable from their differing K_m values. Glucose transporters 1, 3 and 4 have K_m values of ~2–5 mmol/L but GLUT2 has a K_m of ~25 mmol/L. This permits high rates of glucose entry into essential cells (e.g. in the central nervous system (CNS), which is relatively protected from neuroglycopaenia by the low Km of its GLUT3 transporters). Pancreatic β-Cells are able to sense increments in blood glucose over a range considerably exceeding normal values via the high K_m GLUT2 transporter protein.

Certain glucose transporters (GLUT1 and GLUT3) are present on cell surfaces at all times. In contrast, GLUT4 is stored in the cytoplasm when insulin is not present; it responds to insulin by moving from intracellular stores to cell membranes, thereby increasing total transporter number (typically 6–10-fold). When insulin concentrations decline, GLUT4 is removed from cell membranes by endocytosis and rapidly recycled back to its intracellular storage compartments. Erythrocytes are insulin non-responsive because they possess only GLUT1. Many cells can express a variety of different GLUT transporters, and the expression of GLUT receptors changes with circumstances: for example liver cells express more GLUT1 and GLUT3 during starvation. Dysfunction of the insulin-regulated GLUT4 translocation process appears to play a part in the insulin resistance syndrome, and mutations of several transporters (e.g. GLUT1, GLUT2) have been associated with inherited metabolic diseases of carbohydrate metabolism.

Tissue glucose uptake via GLUT transporters involves facilitated diffusion down a concentration gradient, the intracellular concentration of glucose being very low because of its active metabolism by phosphorylating enzymes (hexokinases and glucokinases). In resting, postabsorptive subjects, ~ 70% of the body’s glucose metabolism occurs independently of the action of insulin. However, these insulin-independent mechanisms cannot maintain normoglycaemia for very long without insulin orchestrating the response to food and integrating the balance between fatty acid, ketone body and glucose metabolism. Insulin-independent (as well as insulin-dependent) glucose clearance is impaired in subjects with type 2 diabetes and also in normoglycaemic subjects with a family history of diabetes. This suggests that abnormalities in insulin-independent glucose disposal manifest at a very early stage of disease development. This phenomenon of ‘glucose resistance’ appears to be quantitatively important: in normal subjects, as much as half an intravenous glucose load is cleared by non-insulin-mediated glucose disposal. Although polymorphisms of GLUT4 do not appear to be any more common among patients with type 2 diabetes than in the normal population, there is evidence that targeting and trafficking of this glucose transporter may be abnormal in both skeletal muscle and adipose tissue in this condition. The insulin-sensitizing agents metformin and the thiazolidinediones appear to increase cell surface expression of GLUT4, as does physical exercise.

The ability to move glucose against a concentration gradient, necessary in the special circumstances of the renal tubules and intestinal epithelium, is conferred by a family of sodium-dependent glucose cotransporters. Sodium-dependent glucose cotransporter 1 (SGLT1) is present in the gut, where it is responsible for absorption of glucose and galactose from the diet. Mutations of SGLT1 are associated with the glucose–galactose malabsorption syndrome that may cause fatal infantile diarrhoea unless these sugars are removed from the diet. Sodium-dependent cotransporter 2 (SGLT2) is a low affinity high capacity transporter that is present in the S1 segment of the proximal renal tubules and is responsible for 90% of renal glucose reabsorption, the remaining glucose being reabsorbed by the high affinity but low capacity SGLT1 transporter found in the S3 segment; reduced function mutations of SGLT2 are associated with renal glycosuria. Drugs that block SGLT2 (thus increasing urinary glucose loss) have recently been licensed in the UK as glucose-lowering agents.

In order to trap glucose within cells (since all the GLUTs are potentially bidirectional), glucose is phosphorylated on entry by a family of hexokinases (a). Hexokinase types I–III are widely expressed and have low K_m, but hexokinase type IV (also called glucokinase, and predominantly expressed in liver and the β-Cells of the islets of Langerhans) has a much higher K_m (up to 15 mmol/L), permitting it to function as a glucose sensor over (and beyond) the physiological range of blood glucose. Since glucokinase is also the rate-limiting step in glucose metabolism, it thus becomes the ultimate determinant of the rate of insulin secretion from β-Cells.

Loss of function mutations of glucokinase are responsible for one form of maturity onset diabetes of the young (MODY 2, which represents approximately 30% of all cases of MODY). Patients present with mildly elevated blood glucose concentrations owing to apparent elevation of the set point for blood glucose concentrations. For reasons that are not totally clear, patients with this condition appear to be at very low risk for the development of diabetic complications and are rarely symptomatic. Although a good response is typically obtained with augmentation of the insulin response by sulfonylurea therapy, this is often not necessary. Although glucokinase might be a potential candidate gene in type 2 diabetes (and a treatment target, given that activating mutations can cause hypoglycaemia in man), mutations appear to be no more common among people with type 2 diabetes than in the general population.

Dephosphorylation of glucose (the reverse reaction) is catalysed by glucose 6-phosphatase (b). This process is necessary for the export of glucose (from gluconeogenesis) by hepatic and renal cells in hypo-insulinaemic situations. Deficiency of this enzyme (or of the associated glucose 6-phosphate transport protein) is the cause of glycogen storage disease type 1 (von Gierke disease), and its overactivity is a feature of the increased and relatively insulin-insensitive hepatic glucose production typical of type 2 diabetes. The insulin sensitizing agents metformin and the thiazolidinediones appear to reduce the activity of glucose 6-phosphatase, although it is not clear whether these are direct effects or are mediated through some other upstream action.

Having entered the cell and undergone phosphorylation, glucose undergoes one of four distinct metabolic fates. It may be:

• metabolized anaerobically to yield energy

• used to synthesize other molecules (glycerol and the carbon skeletons of certain non-essential amino acids)

• stored as glycogen.

The dominant flux along each pathway will depend on the tissue (e.g. gluconeogenesis can take place in the liver and kidneys, glycogen storage in liver and muscle, glycerol generation in adipose tissue) and on the prevailing metabolic circumstances such as cellular ATP requirements (requiring glycolysis), ‘housekeeping’ requirements (e.g. plasma membrane stability in erythrocytes), oxygen availability (determining whether glycolysis is anaerobic (yielding lactate) or aerobic (yielding pyruvate, which can be converted to acetyl-CoA and enters the tricarboxylic acid cycle) and alternative substrate availability (e.g. fatty acids or ketone bodies in the fasting state). Some of these pathways, and in particular the rate of glucose oxidation, have been shown to be abnormal in subjects with type 2 diabetes and also in their non-diabetic relatives.

Insulin is a peptide hormone (51 amino acids arranged in two peptide chains linked by two disulphide bonds, molecular weight 5807 Da), which is secreted by the β-Cells of the islets of Langerhans in the pancreas. In the synthesis of insulin, translation of mRNA yields preproinsulin, a prohormone that undergoes post-translational modification prior to release of the mature insulin molecule. Removal of 24 amino acids from preproinsulin yields proinsulin, which consists of 86 amino acids. Proinsulin is stored in secretory granules prior to release from β-Cells by exocytosis. In healthy subjects, > 90% of proinsulin is converted to mature insulin by the removal of the metabolically inert C-peptide component prior to secretion. The other products of the post-translational modification are either released when exocytosis occurs or are degraded within the secretory granules prior to release. C-peptide is co-secreted in equimolar amounts with mature insulin. In healthy subjects, only small amounts (< 10% of mature insulin output) of proinsulin and partially split proinsulin are released. These ratios are characteristically disturbed in certain pathological states, including autonomous insulin secretion from an insulinoma and type 2 diabetes mellitus. Plasma concentrations of proinsulin and C-peptide are low or undetectable in cases of surreptitious administration of exogenous insulin. Assay of these substances may, therefore, prove helpful in the differential diagnosis of hypoglycaemia in some circumstances. Proinsulin may accumulate in renal failure and its plasma concentration is elevated in familial hyperproinsulinaemia.

Substances stimulating the synthesis and storage of insulin include glucose, mannose, leucine, arginine and a variety of metabolizable sugars and sugar derivatives. Most of these also promote secretion.

The mechanisms by which insulin release is triggered are the focus of much research. It is apparent that there is an ATP-dependent, sulfonylurea-sensitive K⁺ channel whose closure is a late event in the intracellular signalling mechanism within the β-Cell; closure triggers calcium influx and exocytosis. It is not clear how this K⁺ channel is activated, although a wide range of secretagogues can stimulate activation of this final common pathway. The most important of these is hyperglycaemia, although mannose, lactate, arginine, leucine and other amino acids, glucagon, glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP, known formerly as gastric inhibitory peptide), cholecystokinin, vasoactive intestinal peptide (VIP), sulfonylureas and parasympathetic cholinergic (muscarinic) nerve activity also stimulate insulin release; many of these secretogogues have synergistic effects. Both neural sympathetic tone and circulating catecholamines inhibit insulin secretion.

More common abnormalities of insulin secretion involve loss of the normal pulsatility of insulin release, an early feature of both obesity and type 2 diabetes. The progressive loss of insulin secretory capacity in diabetes is discussed below.

At present, only two receptors that mediate the actions of insulin have been identified: the insulin receptor and the IGF (insulin-like growth factor) receptor. It is apparent, however, that there are individual dose–response curves for the different actions of insulin in different tissues. For example, the median effective dose (ED₅₀) for insulin’s antilipolytic action on adipose tissue is < 140 pmol/L (20 mU/L) (and may be < 70 pmol/L), while those for inhibition of hepatic glucose output and stimulation of glucose uptake into skeletal muscle are 210–350 pmol/L (30–50 mU/L) and 350–490 pmol/L (50–70 mU/L), respectively. A doubling of insulin concentration inhibits hepatic glucose output by ~80% and stimulates peripheral glucose utilization by ~ 20%. In patients with type 2 diabetes, these differential effects on lipolysis, hepatic glucose output and glucose uptake, coupled with ongoing (if reduced) insulin secretion, are probably responsible for the fact that most individuals are not at risk of developing ketoacidosis (at least for many years), despite the clear defect in glucoregulation that is evident.

The different actions of insulin have different time courses, with the glucoregulatory and antilipolytic occurring within a few minutes, and growth regulation and actions dependent upon synthesis of new proteins occurring over periods of hours or days. Intravenous injection of insulin typically has little effect on blood glucose for 5–10 min, the maximal hypoglycaemic action occurring after 5–15 min. Insulin stimulation of skeletal muscle glucose uptake declines with a half-life of 10–20 min after the insulinaemic stimulus has been removed.

Proinsulin and partially split proinsulins have metabolic activities generally similar to those of insulin, although their plasma half-lives are 3–5 times longer and their biological potencies are only 8–15% that of insulin. It has been suggested that the hepatic activity of proinsulin may be relatively more potent than its effect on peripheral glucose uptake.

The insulin receptor gene is located on the short arm of chromosome 19 (19p 13.2). Rare gene mutations have been described, for example leprechaunism and Rabson–Mendenhall syndrome, which result in severe glucose intolerance with resistance to exogenous insulin, and profoundly disordered growth, rather than typical insulin resistance. These conditions are usually lethal in infancy and adolescence, respectively. There are also more common, ‘milder’ polymorphisms of the insulin receptor gene. However, these appear to explain only a small proportion of the marked variance in population insulin sensitivity and they are considered to be a rare (< 1%) cause of type 2 diabetes mellitus. Indeed, most mutations of the insulin gene that have been recognized are not sufficient to cause diabetes alone, although they may render it more likely to occur in the presence of other risk factors.

Insulin can have multiple actions, even on a single responsive cell, and hence there are probably several different intracellular pathways mediating its actions. Glucoregulatory and antilipolytic responses are rapid and probably mediated via serine and threonine kinases and cAMP. Stimulation of lipid and protein synthesis, inhibition of proteolysis, the nuclear transcription of RNA and the replication of DNA are slower and have different second messenger systems. Second messenger systems involving diacylglycerol, protein kinase C and glycosyl-phosphatidyl-inositol are under investigation, but abnormalities in them have yet been demonstrated to be necessary or sufficient to cause diabetes in man. As a result of these second messenger cascades, glucose transporter proteins are translocated from within cells to their surface membranes to increase glucose flux into the cytoplasm.

After activation, insulin–receptor complexes are internalized by an endocytotic process; receptors are later recycled to the cell surface. Internalization of the insulin is important (and possibly essential) for insulin signals to reach the nucleus and influence cell growth and protein synthesis. Internalization is an important route by which insulin is cleared from the circulation and degraded. The actions of insulin in stimulating DNA transcription and mRNA translation do not depend upon the plasma membrane insulin receptor kinase activity and the second messenger systems discussed above, or on the IGF receptors described below, but rather involve direct effects within the nucleus and on ribosomes.

In addition to its acute effects on glucose uptake and release and on lipid metabolism, insulin has growth promoting activity in a variety of tissue culture models. At least two protein hormones, insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), have actions that partly resemble these actions of insulin. The amino acid sequences of these proteins and the base sequences of their coding DNA are known and show some homology with those of insulin. Insulin-like growth factors are weak agonists for the insulin receptor and hence have weak glucoregulatory and antilipolytic effects. In addition, they have growth-promoting effects mediated by two IGF receptors. Insulin itself binds only weakly to IGF receptors.

The physiological role of IGFs is not yet fully established. It has been suggested that they act as somatomedins (they were previously called somatomedins C and A, terms that are no longer recommended), in that they are induced by growth hormone and mediate its growth-promoting effects in children. In adults, they are believed to promote the growth of continually dividing cells such as osteoblasts, chondrocytes, fibroblasts and erythroid cells.

Diabetes is the most common metabolic disorder, with a cumulative incidence of 5–10% in people in the developed world aged > 40 years, in whom > 90% of cases are due to type 2 diabetes. Population screening programmes typically reveal that up to half of the subjects found to have type 2 diabetes had previously been undiagnosed. While the burden of type 2 diabetes is increasing exponentially with the epidemic of obesity in many parts of world, the incidence of type 1 diabetes has also been increasing for many years for reasons that are much less apparent. Nevertheless, the prevalence of type 2 diabetes in children is approaching that of type 1 diabetes, having been only ~2–4% of all childhood diabetes prior to 1994, and is predicted to outstrip type 1 diabetes by 2025 on current trends. A high proportion of these children, typically presenting around the time of puberty, require insulin from the time of diagnosis. Whether this is due to the accelerator hypothesis (‘double diabetes effect’), whereby an individual’s risk of contracting type 1 diabetes is increased by the prior existence of type 2 diabetes or insulin resistance, is not clear.

Although insulin has many actions, as described above, diabetes is defined only in terms of elevated blood glucose concentrations. Since blood glucose is a continuous variable, the cut-off points for diagnosis are necessarily somewhat arbitrary.

Recent changes to the diagnostic criteria for diabetes and glucose intolerance reflect the increased cardiovascular risk evident at even moderate levels of fasting hyperglycaemia (~6.0 mmol/L in some studies). However, the blood glucose threshold for this effect is almost certainly lower than that for the predominantly microvascular complications unique to diabetes mellitus. Some people so diagnosed may not, therefore, necessarily be at risk of developing the unique set of complications (nephropathy, retinopathy, neuropathy) that have traditionally characterized the disease and determined its management.