F. Belfiore, S. Iannello
Institute of Internal Medicine, University of Catania, Ospedale Garibaldi, Catania, Italy
Pancreatic P-cells synthesize a large polypeptide chain, the proinsulin, which is then cleaved into the so-called connecting peptide (C-peptide) and the insulin molecule, composed of two peptide chains containing 51 amino acid residues. Both insulin and C-peptide are packaged in the secretory granules. During the secretory process, the granule content is discharged outside the P-cell through a process of exocytosis, leading to the release of insulin and C-peptide in equimolar amounts, together with small quantities of uncleaved proinsulin. In contrast to insulin, C-peptide is not taken up by the liver (and the other insulin-sensitive tissues), and therefore its plasma level is a good index of insulin secretion.
Regulation of Insulin Secretion by Substrates Glucose
Glucose is the main physiological regulator of insulin secretion. In vitro, prolonged stimulation with glucose (or sulfonylureas) induces a biphasic insulin secretory response by pancreatic islets characterized by an initial rapid first phase lasting about 5 min, during which about 2-3% of the insulin content of pancreas is released, followed by a slower second phase in insulin secretion, which results in the liberation of up to 20% of total pancreatic content during a period of 60 min of glucose perfusion. A similar biphasic pattern of secretory response to glucose has also been reported in vivo in man with the hyperglycemic clamp technique. The two secretory phases, however, are not apparent after a carbohydrate-rich meal because the elevation in blood glucose is not rapid enough. Nevertheless, an efficient initial insulin secretory response (dependent upon the P-cell sensitivity to glucose elevation) is required for an optimal glucose control and for avoiding an excessive secretion during the second phase, which entails the risk of late hypoglycemia (reactive hypogly-cemia).
Glucose, besides its direct stimulation of insulin release, also potentiates the secretory response to nonglucose stimuli, which may play a role during the absorption of mixed meals. In addition, glucose exerts a priming effect of P-cells, as a previous exposure of P-cells to glucose causes an enhanced secretory response to a subsequent stimulation with glucose (or even with nonglucose stimuli), as if the P-cell has memory of the previous glucose exposure. Chronic exposure to glucose, however, induces desensitization of P-cells, which does not seem to be due to a reduced content or synthesis of insulin. This is relevant to the condition of persistent hyperglycemia occurring in the diabetic state.
With regard to insulin secretion, three concepts should be distinguished: the set point for blood glucose, the P-cell threshold for glucose, and the P-cell glucose sensor. The set point entails the concept that there is a control system that 'sets' the level of glucose at a given value, which in man is fixed to about 5 mmol/l glucose. The set point is the result of the activity of P-cells as well as of a-cells and S-cells.
The glucose threshold for both P-cells and a-cells is between 5 and 6 mmol/l: when glucose rises above this level, the insulin-secreting P-cells are turned on whereas the glucagon-secreting a-cells are turned off, and vice versa. Glucose threshold increases during starvation, when P-cells are blind to even relatively high glucose levels, and returns to normal upon refeeding. In order to be able to respond to increase in glucose concentration above the threshold value, P-cells must be equipped with a glucose sensor, which has been identified in the glucose-phosphorylating enzyme glucokinase (GK). This enzyme, long known to be present in the liver, has been shown to occur also in the P-cells (the liver and the P-cell enzymes differ at genetic level). GK differs from the ubiquitous enzyme hexokinase (which catalyzes the same reaction as GK, i.e. glucose phosphorylation), in that hexokinase has a high affinity (or a low Km) for glucose and therefore works at the maximum activity at very low, under-physiological glucose concentration, whereas GK has a low affinity (or a high Km) for glucose, which entails that its activity increases with increasing glucose concentration. Its presence in the liver allows this organ to take up glucose when glycemia in the portal vein increases (such as during the absorption period), whereas its presence in the P-cells allows these cells to perceive the increase in blood glucose and to respond with adequate insulin release.
In order to stimulate insulin release, glucose must first be transported into the P-cell by the glucose transporter (GLUT-2 isoform), and then phos-phorylated by GK to produce glucose-6-P. However, glucose transport in P-cells possesses a very high capacity and therefore plays a little regulatory role. Glucose-6-P produced by GK is further metabolized along several pathways, through which ATP is generated. Shortly, glucose metabolism results in elevation of the ATP/MgADP ratio which inhibits ATP-sensitive K+-channels, thus lowering membrane potential and triggering Ca influx through the voltage-dependent Ca2+-channels, which stimulates insulin secretion (fig. 1). Genetic alterations of key components of the insulin secretory machinery have been described. Mutations of KATP-channels or the associated sulfonylurea receptors may cause hyperinsulinemia and hypoglycemia due to persistent depolarization of the P-cell membrane. Mutations in the GK gene (most of which affect the glucose-binding site) may result in hyporesponsiveness to glucose, as it occurs in MODY-2 patients, or in hyperresponsiveness, as noted in the familial GK-linked hyperinsulinemia and hypoglycemia (FHI-GK).
Oscillations in the glycolytic pathway and P-cell metabolism contribute to the oscillatory nature of P-cell ionic events and insulin secretion. Insulin release is a complex oscillatory process with rapid pulses (10 min) superimposed on slower circhoral oscillations (50-100 min). Moreover, ultradian oscillations of insulin secretion appear to be an integral part of the feedback loop between glucose and insulin secretion, and are abnormal in states of glucose intolerance.
Fats also influence insulin release. In man, FFA were shown to enhance the secretory response to glucose, which is in agreement with the demonstration that pancreatic islets are equipped with the enzymes necessary for the utilization of FFA and ketone bodies. Amino acids such as isoleucine, arginine and lysine, potentiate the secretory effect of glucose, whereas leucine may be regarded as a primary stimulus, active even in the absence of glucose. Amino acids do not seem to act by serving as fuels for P-cells. They might act by contributing to activate Ca channels.
An important signal for insulin secretion may reside in the inextricable interplay between glucose and lipid metabolism. Specifically, glucose metabolism leads to the generation of malonyl-CoA, which inhibits carnitine palmi-toyltransferase-1, with the attendant accumulation of long-chain acyl-CoA esters in the cytosol (see also chapter III and figure 3). Malonyl-CoA and long chain acyl-CoA esters may act as metabolic coupling factors in P-cell signalling.
Fig. 1. Regulation of insulin secretion by the p-cell. (Continuous lines ending with black arrows indicate transformation or translocation of substrates or ions; dotted lines ending with white arrows indicate stimulation; dotted lines ending with filled circles indicate inhibition). Glucose metabolism (regulated by GK which acts as 'glucose sensor') results in production of ATP which inhibits ATP-sensitive K+-channels, thus lowering membrane potential and triggering Ca influx through the voltage-dependent Ca2+-channels. High cytosolic Ca stimulates (through complex processes, not shown) insulin secretion. Sulfonylureas stimulate insulin secretion by acting through their receptor, closely associated with the ATP-sensitive K+-channels. Parasympathetic stimulation (acetylcholine) promotes insulin secretion through activation of PLC, which produces IP3 and DAG (from PIP2); IP3 causes release of Ca from the intracellular stores (endoplasmic reticulum) into cytosol; DAG activates PKC which in turn stimulates secretion. Glucagon enhances secretion by activating AC (with the participation of Gs), thus producing cAMP and activation of PKA. Epinephrine (through the a2-receptor) inhibits secretion by inhibiting AC (with the participation of Gi), thus exerting effects opposed to those of glucagon.
Abbreviations (alphabetic order): a2 = a2-Adrenergic receptor; AC = adenylate cyclase; cAMP = cyclic AMP; DAG = 1,2-diacylglycerol; GK = glucokinase; Glg = glucagon receptor; GLP1 = glucagon-like peptide 1; Gq = a further type of G protein; Gs and Gi = stimulatory and inhibitory G proteins; IP3 = inositol-1,4,5-trisphosphate; M3 = a type of muscarinic receptor; PIP2 = phosphatidylinositol-4,5-P; PKA = protein kinase A; PKC = protein kinase C; PLC = phospholipase C.
Regulation of Insulin Secretion by Hormones and Neurotransmitters
Acetylcholine, produced by parasympathetic activity, stimulates insulin secretion through muscarinic receptors (which can be blocked by atropine), probably by enhancing DAG (diacylglycerol) and IP3 (inositol-3-P) formation (fig. 1). Parasympathetic stimulation may occur during the early (cephalic and intestinal) phase of insulin secretion following a meal as well as during hypoglycemic episodes. In the latter instance, however, hypoglycemia limits the parasympathetic effect on insulin secretion, because this effect is glucose-dependent. The parasympathetic innervation of the pancreas may also trigger the release of vasoactive intestinal polypeptide (VIP), which stimulates the secretion of insulin (and glucagon) while increasing the blood flow to the pancreas and the external pancreatic secretion.
Norepinephrine (released upon sympathetic stimulation) and epinephrine (produced by adrenal medulla) exert both an inhibitory effect, through the a-adrenergic receptors (fig. 1), and a stimulatory effect, through the P-adrenergic receptors, the overall effect being an inhibition of glucose-stimulated insulin release and a little effect in the basal state. Sympathetic nerve activity may also release other neurotransmitters, such as galanin, which would inhibit both basal and stimulated insulin secretion.
Gastrointestinal hormones (or gut hormones) contribute to the overall insulin secretion, as shown by the higher insulin secretion after glucose given per os compared to intravenous glucose. For this action, they are also called incretins. They include: the gastric inhibitory polypeptide (GIP), secreted by the endocrine cells of duodenum and jejunum; cholecystokinin (CCK), both the long (CCK-33) and the short (CCK-8) peptide chain, released by duodenum and proximal part of jejunum after ingestion of fats and proteins; the glucagon-like peptide-1 (7-36) amide, or GLP-1 (7-36), formed from GLP-1 (the precursor proglucagon, produced by the L-cells in the distal part of small intestine, is processed by tissue-specific proteolysis to produce glucagon in pancreatic a-cells and GLP-1 in the intestine), is released after carbohydrate-rich meals (fig. 1); the neuropeptide Y (NPY), a neurotransmitter present in both the central nervous system and the enteric nervous system which produces stimulation of food intake (and of resting metabolic rate), while probably acting as an incretin to enhance insulin release.
The counterregulatory hormones (or stress hormones) also affect insulin secretion. Glucagon is a potent stimulus for the islet P-cell (fig. 1), and intravenous bolus injection of 1 mg glucagon has been widely used to assess endogenous insulin secretion for clinical or research purposes. Glucagon stimulates insulin release mainly through glucagon receptors but not GLP-1 receptors on islet P-cells. On the other hand, insulin may affect glucagon secretion because capillaries go from the central part of the islets, where insulin is mainly produced, to the periphery of the islets where glucagon-producing a-cells are mainly located. The other stress hormones affect insulin secretion through a generally inhibitory action. The effect of epinephrine has been mentioned above. Cortisol and growth hormone (GH) are thought to play a role during prolonged stress periods.
Leptin may potentiate glucose-induced insulin secretion by a mechanism involving cAMP or phospholipase C/protein kinase C activation. It also inhibits NPY release. In contrast to early studies, recent data indicate that amylin is a third active pancreatic islet hormone that works with insulin and glucagon to maintain glucose homeostasis. It would regulate glucose inflow to the circulation by influencing the rate of gastric emptying and would also inhibit hepatic glucose production in the postprandial period.
Fasting Insulin Level. The fasting insulin level (normally between 5 and 15 |iU/ml) may reflect the insulin secretory capacity. It may be very low ( = 5 |iU/ml) in subjects with high insulin sensitivity (lean and/or trained subjects) and elevated (>15 ^U/ml) in insulin-resistant subjects. It should be pointed out that an apparent normal insulin level in insulin-resistant diabetic subjects indicates decreased secretory capacity, since an equal level of glucose in a 'normal' subject would be associated with a higher insulin level which would promptly normalize glucose.
It should be pointed out that the insulin values usually referred to are those obtained with the commonly used radioimmunoassay method, which yields the total insulin levels, whereas more sophisticated methods are available that allow to distinguish the true insulin from the proinsulin. True insulin may be lower than total insulin by 15-20%.
Acute Insulin Response to Glucose (AIRG). AIRG following glucose given as intravenous bolus consists of a rapid increase in insulin level which returns towards normal within 10 min. The magnitude of AIRG is not affected by the preexisting glucose level, which makes this test feasible even in diabetic patients. AIRG is often absent in patients with type 2 diabetes whereas it is enhanced in insulin-resistant obese subjects.
Acute Insulin Response to Non-Glucose Stimuli (AIRNG). AIRNG includes response to amino acids, neurotransmitters and hormones. AIRNG obtained with arginine increases with the increase in the preexisting glucose level. By plotting the AIRNG values against those of glycemia, a 'curve' is obtained which reflects the correlation between these two variables. From the analysis of this curve, it is possible to deduce the insulin secretory capacity of the pancreas, the maximal acute insulin response to nonglucose stimuli or AIRmax , and the P-cell sensitivity to the potentiation effect of glucose. The AIRmAx (which indicates the maximal secretory response) is reduced in type 2 diabetes and may increase in insulin-resistant hyperinsulinemic subjects. The P-cell sensitivity to the potentiation effect of glucose is little changed in type 2 patients, suggesting preserved P-cell sensitivity in these patients.
Both impaired insulin action (insulin resistance) and reduced insulin secretion (insulin deficiency) may contribute to the development of type 2 diabetes. It is now accepted that in type 2 diabetes the situation may range from predominantly insulin resistance with relative insulin deficiency to a predominantly secretory defect with insulin resistance. It is noteworthy that recent data would suggest that the hyperinsulinemia of insulin resistance may result from an increase in insulin secretion secondary to increased P-cell sensitivity to glucose, as well as a decrease in insulin clearance. In type 2 diabetes the P-cell mass is reduced by about 50%, which is known from experimental pancreatectomy to be not enough to cause fasting hyperglycemia. Therefore, in most type 2 patients a functional defect in P-cells may occur, leading to insulin secretory defect. This is confirmed by the almost absent acute insulin response to glucose (AIRo), diminished maximal acute insulin response to nonglucose stimuli (AIRmAx), decreased insulin secretory capacity, with normal P-cell sensitivity to the potentiation effect of glucose.
Type 2 diabetes subjects have their own 'set' of fasting plasma glucose which is more or less increased compared to normal. This is due to reduced insulin secretion into portal vein which is unable to completely suppress hepatic glucose production, resulting in hyperglycemia. The latter, on the other hand, is somewhat 'useful' in that it forces the hypofunctioning P-cell to secrete more insulin. The apparently 'normal' fasting insulin in type 2 diabetes in the presence of fasting hyperglycemia should indeed be considered as reduced. In fact, restoring normal glucose levels in mild diabetes by an insulin infusion reduces the endogenous insulin concentration to subnormal values. The same reasoning apply for the insulin response to oral glucose, i.e. to the insulin curve during OGTT. The insulin response (area under the curve or AUC) may be normal or most often elevated in absolute terms, but should be regarded as reduced considering the elevated glycemic values. Moreover, the insulin response during OGTT may show a sluggish initial response and elevated values in the later stages, the latter perhaps resulting from the elevated glucose values. In type 2 diabetes there is also an increased release of proinsulin, which may account for 30% of total insulin compared to 15% in normal subjects.
Concerning the insulin response to intravenous glucose, as occurs during IVGTT, in type 2 diabetes there is a marked reduction in the first phase of insulin release. The second phase may also be reduced in diabetic patients with fasting glycemia > 250 mg/dl, but may be normal or increased in 'compensated' patients with fasting glycemia <200 mg/dl (even if also in these instances the insulin response should be regarded as diminished, considering the existing hyperglycemia). Reduced insulin response is also recorded during prolonged glucose infusion.
The insulin response to nonglucose stimuli, such as intravenous arginine, secretin, isoproterenol, isoprenaline, tolbutamide, or even a mixed meal, may be normal in type 2 diabetic patients with fasting glycemia < 200 mg/dl. This is due to the potentiation of the insulin response to nonglucose stimuli exerted by the hyperglycemia present in the diabetic patients.
Finally, in type 2 diabetic patients the oscillations in insulin secretion, which are significant for glycemic control, cannot be detected, even in the patients with mild form of the disease.
A major role is certainly played by genetic predisposition, but several biochemical mechanisms and neurohormonal factors may contribute. Little is known about susceptibility genes to the common polygenic forms of type 2 diabetes. Studies of genes involved in insulin secretion or insulin action have been successful to a certain extent by showing the implication of the insulin-receptor substrate-1 (IRS-1) gene, the ras associated with diabetes (rad) gene, the glucagon receptor gene, or the sulfonylurea receptor (SUR) gene (among others) in a low percentage of cases of type 2 diabetes in particular populations. However, the majority of susceptibility genes are still to be described.
Recently, an inherited or acquired defect of FAD-linked mitochondrial glycerophosphate dehydrogenase in (3-cells has been proposed to contribute to the impairment of insulin release in type 2 diabetes.
Intravenous administration of (-endorphins or naloxone to type 2 diabetic patients enhances both basal and OGTT stimulated insulinemia, which suggests a possible pathogenetic role of these compounds in the dysfunction of (-cells.
Prostaglandins may also be implicated, as suggested by the improvement of insulin response to intravenous glucose and the increase of the slope of glucose potentiation after infusion of sodium salicylate (inhibitor of prostaglandin synthesis). A similar effect has been observed with the a-adrenergic blocking agent phentolamine, which suggests a role of the a-adrenergic system. It has also been suggested that galanin and pancreostatin, peptides which inhibit insulin secretion, may be increased in the pancreatic islets of type 2 diabetic patients. Finally, hyperglycemia, once established, may contribute to aggravate the ß-cell dysfunction, through several mechanisms most of which are included in the concept of 'glucotoxicity'. The glucotoxicity concept may help to explain the beneficial effect on insulin secretion obtained in type 2 diabetic patients after adequate treatment achieving glycemic control as well as the transient improvement in the ß-cell function which may occur in type 1 diabetic patients after therapeutical control of hyperglycemia ('honeymoon' phenomenon).
It has been proposed that at least one factor contributing to the pathogen-esis of type 2 diabetes is desensitization of the GLP-1 receptor on ß-cells. At pharmacological doses, infusion of GLP-1, but not of GLP, can improve and enhance postprandial insulin response in type 2 patients. Agonists of GLP-1 receptor have been proposed as new potential therapeutic agents in type 2 diabetic patients.
It should also be emphasized that complex alterations of glucidic and lipidic metabolism in the ß-cells may play a role. In particular, in obese/diabetic hyperinsulinemic subjects, LC-CoA derived from the enhanced availability of FFA may affect the ß-cells' secretory response according to the following mechanism: as the glycemic level increases, the ß-cells utilize more glucose; this leads to enhanced production of malonyl-CoA, which blocks the intrami-tochondrial transport of LC-CoA, which therefore accumulates in the cytosol and (through its complex biological effects) stimulates insulin secretion (see also chapter III and figure 3 for details).
Altered expression of genes encoding enzymes in the pathway of malonyl-CoA formation and FFA oxidation contributes to the ß-cell insensitivity to glucose in some patients with type 2 diabetes. Clearly, the detrimental impact of diabetic hyperlipidemia on ß-cell function has been a relatively neglected area, but future pharmacological approaches directed at preventing 'lipotox-icity' may prove beneficial in the treatment of diabetes.
Various, less common types of diabetes are known to occur, in which the secretory defect is based upon different mechanisms, as outlined in chapter I on Etiological Classification.
Pharmacological Stimulation of Insulin Secretion
The main drugs able to stimulate insulin secretion are the sulfonylureas. These compounds have been used in the management of type 2 diabetes since 1955 and, when properly utilized, are easy to use and appear to be effective and safe. It is estimated that 30-40% of diabetic patients are taking oral sulfonylureas. Indications and contraindications for sulfonylureas are shown in tables 1 and 2, respectively.
Table l. Patients candidate for sulfonylurea treatment
Most patients with type 2 diabetes, not well controlled with dietary restriction and exercise Children and adults with the MODY (maturity-onset diabetes of youth) type of diabetes Obese-diabetic patients with marked insulin resistance Lean type 2 diabetic patients with preserved insulin secretory capacity
Table 2. Contraindications to sulfonylurea treatment
Patients with type 1 diabetes Patients with pancreatic diabetes
Patients with an acute illness or stress or undergoing surgery Patients with hepatic or liver diseases Patients predisposed to hypoglycemia: Underweight or malnourished Elderly Diabetic pregnancy: Potential teratogenicity Perinatal mortality Severe neonatal hypoglycemia Diabetic female patients during lactation
Patients with a history of severe adverse reactions to sulfonylureas
The first oral hypoglycemic drug was synthesized in 1926 by altering the guanidine molecule. The sulfonylureas used today are derived from this native molecule. The 'first-generation' sulfonylureas, which were developed initially, are effective in large doses, while the 'second-generation' drugs, developed more recently, are effective in smaller doses. Some sulfonylureas, such as tolbutamide,
Table 3. Main characteristics of sulfonylureas
Compound Dose, mg/day Doses q.d. Duration of Metabolism/
hypoglycemic excretion effect, h
Compound Dose, mg/day Doses q.d. Duration of Metabolism/
hypoglycemic excretion effect, h
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