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Sharpey-Schafer Lecture |
1 School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, UK
Abstract
Knowledge about the sites and actions of the numerous physiological and pharmacological factors affecting insulin secretion and pancreatic ß-cell function has been derived from the use of bioengineered insulin-producing cell lines. Application of an innovative electrofusion approach has generated novel glucose-responsive insulin-secreting cells for pharmaceutical and experimental research, including popular BRIN-BD11 ß-cells. This review gives an overview of the establishment and core characteristics of clonal electrofusion-derived BRIN-BD11 ß-cells. As discussed, BRIN-BD11 cells have facilitated studies aimed at dissecting important pathways by which nutrients and other bioactive molecules regulate the complex mechanisms regulating insulin secretion, and highlight the future potential of novel and diverse bioengineering approaches to provide a cell-based insulin-replacement therapy for diabetes. Clonal BRIN-BD11 ß-cells have been instrumental in: (a) characterization of KATP channel-dependent and -independent actions of nutrients and established and emerging insulinotropic antidiabetic drugs, and the understanding of drug-induced ß-cell desensitization; (b) tracing novel metabolic and ß-cell secretory pathways, including use of state-of-the-art NMR approaches to provide new insights into the relationships between glucose and amino acid handling and insulin secretion; and (c) determination of the chronic detrimental actions of nutrients and the diabetic environment on pancreatic ß-cells, including the recent discovery that homocysteine, a risk factor for metabolic syndrome, may play a role in the progressive demise of insulin secretion and pancreatic ß-cell function in diabetes. Collectively, the studies discussed in this review highlight the importance of innovative experimental ß-cell physiology in the discovery and characterization of new and improved drugs and therapeutic strategies to help tackle the emerging diabetes epidemic.
(Received 28 September 2006;
accepted after revision 1 February 2007; first published online 1 February 2007)
Corresponding author N. H. McClenaghan: School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, UK. Email: nh.mcclenaghan{at}ulster.ac.uk
Preface
The Sharpey-Schafer Lecture and Prize is in memory of Sir Edward A. Sharpey-Schafer FRS and his grandson Professor E. P. Sharpey-Schafer FRCP. Sir Edward Sharpey-Schafer was a distinguished professor of physiology in University College London and University of Edinburgh, widely known to the general public for introducing a method of artificial respiration, a forerunner to modern cardiopulmonary resuscitation. Professor E. P. Sharpey-Schafer was Professor of Medicine, St Thomas' Hospital London and made important contributions to cardiovascular and respiratory physiology, including the understanding of the effects of respiratory acts on circulation.
Sir Edward Sharpey-Schafer was born Schäfer in London in 1850, entered University College London in 1868, and in 1871 was the first recipient of the Sharpey Scholarship at University College. At University College, he took the name Sharpey-Schafer as a mark of respect to William Sharpey who helped define his career path in physiology, and was awarded the second Jodrell Professorship of Physiology in 1883. While Sir Edward Sharpey-Schafer is arguably best known for his pioneering studies on the effects of adrenaline on blood pressure and the autonomic nervous system (1894–1895), he made a number of other important contributions to physiology, receiving international recognition for his work on endocrine gland secretions in the 1890s.
Indeed, Sir Edward was one of the founders of endocrinology, and made great contributions to the development of endocrinology as a discipline. Notably, Sir Edward suggested in 1895 that the pancreatic islets may function as a gland that regulates blood sugar, and from 1913 he popularized the term ‘insuline’ for the as yet unidentified pancreatic secretion controlling blood sugar. Later, in 1921, Dr Frederick Banting and Dr Charles Best (working under Professor John MacCleod) isolated a substance they called ‘isletin’ from dog pancreas. As legend has it, Scotsman and friend of Sharpey-Schafer, MacCleod was on holiday when Banting and Best named the substance isletin and on his return he strongly suggested they adopted the name ‘insulin’ as a mark of respect to Sharpey-Schafer and Jean de Meyer, who first coined the term. The landmark discovery of insulin had an immediate impact on the treatment of diabetes, highlighting the importance of the pancreatic islets and constituent insulin-secreting pancreatic ß-cells in the physiological control of circulating blood glucose.
Introduction
The pancreas plays a key role in the metabolism of carbohydrates, proteins and lipids (see McClenaghan & Flatt, 2003; Pickup & Williams, 2003). The endocrine function of the pancreas resides within the pancreatic islets of Langerhans, small microscopic cell clusters suspended in exocrine tissue and constituting 1–3% of the total pancreatic mass. Pancreatic islets are comprised of four major types of cell: insulin-producing ß-cells, glucagon-secreting 
-cells, somatostatin-producing 
-cells and pancreatic-polypeptide-producing PP-cells (see Pickup & Williams, 2003). As illustrated in Fig. 1, rodent islets are typically composed of a central core of ß-cells surrounded by an outer mantle of other cell types which lie in close proximity and interact to regulate endocrine islet secretions. However, as recently noted, human islet architecture does not typically show anatomical subdivision, with different islet hormone-containing cells scattered throughout the islet, suggesting enhanced paracrine interactions in human islets (Cabrera et al. 2006). A wide range of nutrients, hormones and neurotransmitters influence the secretion of insulin and other islet hormones, and the major physiological regulator of pancreatic insulin secretion and ß-cell function is the prevailing concentration of plasma glucose (see Flatt & Lenzen, 2004).
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Understanding of the pathogenesis and aetiology of diabetes relies on unravelling of the complex mechanisms underlying insulin secretion and action, the interplay between ß-cells and insulin-sensitive tissues, and the demise of the pancreatic ß-cell coupled with declining insulin sensitivity (McClenaghan, 2005). While there has been much debate concerning the relative contribution of impaired insulin secretion or insulin action to the onset and pathogenesis of diabetes, it is clear that insulin secretion is defective at the time of diagnosis and declines with type 2 diabetes progression, often paralleled with worsening insulin sensitivity (see Pickup & Williams, 2003; McClenaghan, 2005). The following sections represent an overview of my research to date, which has focused on the physiological and pharmacological regulation of the pancreatic ß-cell with the aim of providing novel functional insights for the understanding and therapy of diabetes.
Bioengineering pancreatic ß-cells
Numerous physiological and pharmacological factors affect insulin secretion, highlighting the considerable progress made over the past four decades in the understanding of pancreatic ß-cell function (Pickup & Williams, 2003; Flatt & Lenzen, 2004). Many of the insights into the mechanisms regulating insulin production and secretion have been derived from studies of freshly isolated pancreatic islets and constituent ß-cells or bioengineered insulin-secreting cell lines (McClenaghan & Flatt, 1999a,b). Considerable effort has been devoted to the production of transplantable and inheritable insulinomas and insulin-producing cell lines, with the principal aim of producing large quantities of functional tissue for experimental ß-cell research.
Provision of insulin-secreting cell lines overcomes many of the shortcomings associated with the use of freshly isolated islets and constituent cells, including the difficulties of preparing large amounts of functional islets, cellular and hormonal heterogeneity, and rapid functional decline in tissue culture (McClenaghan & Flatt, 1999a). While early cell lines, such as RINm5F and HIT-T15, are a rather crude proxy for normal ß-cells, the advances in molecular biology and emergence of novel bioengineering technologies have provided new opportunities to improve and establish more appropriate cultured cell lines (McClenaghan & Flatt, 1999a). Indeed, bioengineered pancreatic ß-cell lines have helped facilitate studies of the mechanisms of insulin secretion, ß-cell dysfunction and destruction, and identification and characterization of drug targets and actions, and have paved the way towards future cell-based therapies for type 1 diabetes.
Establishment of clonal hybrid glucose-responsive ß-cells by electrofusion. Since its first introduction in 1909 by Küster, cell-to-cell fusion has proven a valuable tool in membrane research, genetic mapping and genetic engineering. Fusion mediated by chemicals and inactivated viruses has led to new strains of bacteria, yeasts, fungi, hybrid plants and hybrid mammalian cell lines (see review by Zimmermann, 1986). The severe limitations of conventional approaches led Zimmermann and colleagues to develop an electrical fusion technique. Electrofusion involves bringing normal and fusion partner cells in suspension into tight membrane contact using an AC electrical field, and cell pairs or multiples are fused by application of short high-intensity DC pulses. This results in controlled, reversible membrane breakdown in localized zones and cell fusion. Since electrofusion provides efficient control of the fusion process and facilitates high yields of viable cells, it represents a particularly effective way to establish novel hybrid cell lines.
Novel electrofusion-derived hybrid insulin-secreting cell lines offer many merits, in that they should be able to be grown up in very large numbers and, with long-term stability in tissue culture, offer potentially useful model ß-cells. We theorized that fusing normal pancreatic ß-cells with a cultured cell line, such as RINm5F, would provide a source of novel ß-cell clones. As such, electrofusion-derived hybrid cells should obtain immortality from the RINm5F fusion partner and intact features of insulin biosynthesis and secretion from the normal parental pancreatic ß-cells. Adopting this novel approach, three clonal rodent cell lines were established following electrofusion of New England Deaconess hospital (NEDH) rat ß-cells with immortal RINm5F cells, originally derived from the transplantable NEDH rat insulinoma (McClenaghan et al. 1996a,c). Procedures adopted for the isolation of these three clonal ß-cells (denoted BRIN-BD11, BRIN-BG5 and BRIN-BG7) are described in detail elsewhere (McClenaghan et al. 1996a,c), but the BRIN nomenclature comes from the B-cell-RIN cell fusion, with each cell line cloned from well B into three wells: D11, G5 and G7. Cultured BRIN cells form monolayers, with an epithelial cell morphology, taking on a pavemental pattern when confluent.
Features of electrofusion-derived clonal pancreatic ß-cells. Glucose is the principal physiological stimulator of insulin secretion, entering the pancreatic ß-cell through membrane-bound glucose transporters (GLUT2 in rodents, GLUT1 in humans). This hexose sugar then undergoes rapid phosphorylation and metabolism by glucokinase to generate ATP, and the increase in intracellular ATP:ADP ratio causes closure of KATP channels (see Tarasov et al. 2004), resulting in membrane depolarization, opening of voltage-dependent calcium channels (VDCCs), a rapid rise in intracellular Ca2+ ([Ca2+]i) and ultimately insulin release (see Flatt & Lenzen, 2004; Wiederkehr & Wollheim, 2006). Inappropriate glucose responsiveness, characteristic of most insulin-secreting cell lines, has been attributed to a number of functional abnormalities relating to glucose transport and metabolism, with the absence or reduced expression of two key elements of the glucose-sensing mechanism, GLUT2 and glucokinase (see reviews by McClenaghan & Flatt, 1999a,b).
Primary characterization of clonal rat BRIN cells revealed expression of GLUT2 in membranes from BRIN-BG5, BRIN-BG7 and BRIN-BD11 cells. Notably, the two enzymes responsible for glucose phosphorylating activity, glucokinase and hexokinase, were also detected in the hybrid BRIN cells. Effective ß-cell glucose-sensing comes from a high glucokinase:hexokinase ratio and, while glucokinase only contributes around 5% to the total glucose phosphorylating activity in RINm5F cells, this was much higher in each of the three BRIN cell clones. With the highest GLUT2 protein expression and a high glucokinase:hexokinase ratio, the BRIN-BD11 cells showed the most impressive glucose-sensing ability (McClenaghan et al. 1996a), which translated into a stepwise increase in insulin release in response to 4.2–16.7 mM glucose (P < 0.01 to P < 0.001). While most insulin-secreting cell lines are either unresponsive, as in the case of parental RINm5F cells, or respond to subphysiological glucose concentrations, the threshold for glucose-stimulated insulin release in BRIN-BD11 cells is as reported in normal ß-cells, and these cells demonstrate a strong first phase insulin release with sustained release at a level marginally higher than basal, corresponding to the second phase (McClenaghan et al. 1996a). The importance of glucokinase:hexokinase ratio to effective glucose-sensing is illustrated by the features of BRIN-BD11 cells (McClenaghan et al. 1996a, 1998b) and through establishment of glucose-stimulated insulin release in RINm5F cells by overexpression of glucokinase (Tiedge et al. 2000).
Physiological regulation of insulin secretion and BRIN-BD11 ß-cell function. In addition to exhibiting physiological aspects of ß-cell glucose-sensing, BRIN-BD11 cells transport and utilize other hexose sugars (McClenaghan et al. 1996d) and a wide range of amino and keto acids (McClenaghan et al. 1996b, 1998b; McClenaghan & Flatt, 1999c, 2000). Consistent with studies on normal ß-cells, BRIN-BD11 cells exhibit appropriate stimulatory responses to the enteroinsular hormones GIP, GLP-1(7–36)amide and CCK-8 and to the acetylcholine analogue carbachol, with inhibitory effects being exerted by the catecholamines adrenaline and noradrenaline (see McClenaghan & Flatt, 1999a,b). These actions demonstrated that BRIN-BD11 cells express functional receptors for these physiological modulators, coupled with intact second messenger systems. Indeed, BRIN-BD11 cells have been characterized in detail, and express many key features of normal pancreatic ß-cells (see McClenaghan & Flatt, 1999a,b), including the two-component KATP channel (Kir6.2 and SUR1), VDCCs, and elements regulating late stages of the insulin secretory pathway, including phospholipase C (PLC)/protein kinase C (PKC) and adenylate cyclase (AC)/protein kinase A (PKA) (Fig. 2A). BRIN-BD11 cells have also been useful in studies probing so-called KATP channel-independent actions of glucose, nutrients and insulinotropic drugs (e.g. McClenaghan & Flatt, 1999b, 2000; Ball et al. 2000a,b, 2004a), and have also been used in investigations of ß-cell dysfunction, demise and destruction (Fig. 2B; e.g. Conroy et al. 2002; Picton et al. 2002; Liu et al. 2006). Notably, BRIN-BD11 cells are phenotypically and functionally stable for over 4 months (50 passages) in culture and, although they do not match the granulation, having less than 5% of the insulin content of primary rat ß-cells, importantly they demonstrate regulated insulin release, consistent with observations on primary ß-cells. With relatively lower granulation, BRIN-BD11 cells may be less useful for in-depth study of the processes underlying exocytosis, although it is unrealistic to expect a bioengineered clonal cell to exactly match a freshly isolated cell. In this regard, however, the many merits evident from the characterization and use of BRIN-BD11 cells in diverse functional studies outlined in this review should be balanced against the short lifespan of primary ß-cells, which also suffer from a rapid decline in function and granulation with time in culture.
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Various experimental studies have provided proof of concept (e.g. Tiedge et al. 2000; Davies et al. 2001) and, as illustrated in Fig. 2C, implantation of bioengineered glucose-responsive BRIN-BD11 ß-cells have been demonstrated to effectively correct diabetic hyperglycaemia, with a pronounced increase in insulin content in vivo (Fig. 2D; Davies et al. 2001). A similar improvement of hyperglycaemia has also been achieved following implantation of glucose-responsive RINm5F cells overexpressing glucokinase (Tiedge et al. 2000), further illustrating the validity of this approach. Collectively, these observations prompt research directed towards successful establishment of glucose-responsive bioengineered human ß-cells, providing a large and much needed source of cultured human ß-cells tissue for experimentation and possible transplantation therapy of type 1 diabetes.
Functional differentiation of islet cells from embryonic stem cells. Another exciting approach to generate insulin-producing cells is through directed differentiation of stem cells to a ß-cell phenotype (see recent reviews by Bonner-Weir & Weir, 2005; Keller, 2005; Madsen, 2005). Several studies have demonstrated that embryonic stem (ES) cells can be differentiated into insulin-expressing phenotypes by transfecting ES cells with a construct that functionally couples the regulatory region of the human insulin gene to one that confers drug resistance, or by spontaneous differentiation by culture under selective conditions. The landmark paper by Lumelsky et al. (2001) sparked an intense wave of activity with some reported successes and, while inherent difficulties in this approach have been highlighted, this has clearly not deterred the very many investigators working in this area. Most differentiation strategies for producing insulin-containing cells from ES cells involve withdrawal of leukaemia inhibitory factor (LIF) from the culture medium, resulting in formation of embryonic bodies, and a key differentiation step relies on ES cell treatment with culture medium supplemented with a cocktail of growth factors and other additions (e.g. Lumelsky et al. 2001).
As shown in Fig. 3A, modifications of the five-stage Lumelsky protocol can generate differentiated mouse ES cells expressing key islet genes and hormones, and the formation of cell clusters (pseudoislets) may provide islet-hormone-secreting cells suitable for transplantation in diabetes. Indeed, primary studies in our group have demonstrated that mouse ES-derived clonal insulin-containing cells are responsive to diverse insulin secretagogues (Fig. 3B). However, there are potentially a large number of growth factors, metabolites, peptides and hormones that may help direct ES cells to islet cell phenotypes. Particular interest has been directed towards the potential of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which are known to enhance ß-cell growth, differentiation, proliferation and survival (Green et al. 2004; Liu et al. 2004). In a recent report, Bai et al. (2005) demonstrated that GLP-1 encouraged growth and differentiation of ES cells into insulin-producing ß-like cells, and another study by our group has demonstrated that the stable GIP analogue, GIP(LysPal16), directed mouse ES cells to differentiate into cells with significantly enhanced insulin production (Marenah et al. 2006). While these initial successes show promise, it remains unclear how closely stem cell-derived cells can be expected to resemble normal ß-cells in terms of gene expression, metabolism, growth potential and secretory function. Concerns have been raised about the absolute yield of insulin-positive cells and their relative insulin content, the fate of non-differentiated cells, and inherent oncogenic risks that need to be overcome before such cells can be used therapeutically. This prompts further research directed to understanding the factors controlling ß-cell differentiation, with the view to successful establishment of functionally competent insulin-producing cells of stem cell origin.
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Cell desensitization is commonly observed in eukaryotic cells and is understood to have an underlying role in cell protection. The phenomenon of cell desensitization has been defined as a temporary, readily induced and reversible state of cellular refractoriness attributed to repeated or prolonged exposure to high concentrations of a stimulus, and has been largely attributed to changes in receptor-mediated events, including modulation of gene expression, ion channels, protein phosphorylation, uncoupling from G-proteins and mitochondrial metabolism. The decline in glucose-lowering ability during long-term sulphonylurea therapy (Karam et al. 1986) and reported glucose-desensitization in diabetes prompted our group to undertake a systematic investigation of the hypothesis that prolonged exposure to insulinotropic antidiabetic drugs could lead to pancreatic ß-cell desensitization (Chapman et al. 1999; Ball et al. 2000a,b, 2004a,b, 2005; McClenaghan et al. 2000, 2001). Indeed, the emergence of the concept of desensitization has attracted considerable interest and is considered relevant to the natural history of type 2 diabetes and loss of efficacy of treatment with oral insulinotropic antidiabetic drugs.
Induction of ß-cell drug-desensitization. Studies of the long-term effects of physiological and pharmacological agents have various inherent complications, including difficulty in interpretion of in vivo data, phenotypic instability of pancreatic islets in vitro, and the relatively short functional lifespan of pancreatic islets in vitro. Such research has been greatly facilitated by cultured insulin-secreting cell lines and, as outlined earlier, clonal BRIN-BD11 cells represent a particularly useful ß-cell model, exhibiting long-term stability in culture coupled with intact responses to glucose and other physiological and pharmacological agents. Indeed, BRIN-BD11 cells have been employed to examine the acute and chronic ß-cell effects of established and emerging insulinotropic antidiabetic drugs, including the sulphonylureas (tolbutamide and glibenclamide) and the phenylalanine-derivative nateglinide.
Both 50–200 µM tolbutamide and nateglinide can initiate potent concentration-dependent insulin-releasing actions at non-stimulatory (1.1 mM) glucose (P < 0.01 to P < 0.001) and, under membrane depolarizing conditions (stimulatory 16.7 mM glucose superimposed upon a high 30 mM concentration of KCl), both agents can elicit significant (P < 0.05 to P < 0.001), concentration-dependent, KATP channel-dependent and KATP channel-independent insulinotropic actions. At least one target moderating the KATP channel-independent actions of insulinotropic drugs is the so-called granular sulphonylurea binding protein (gSUR), which regulates chloride influx into the secretory granules and insulin exocytosis (Fig. 4; Eliasson et al. 2003). As such, it was of interest to examine whether prolonged exposure to sulphonylureas and other classes of antidiabetic drugs could result in desensitization of KATP channel-dependent and/or KATP channel-independent insulin-secretory actions of these agents. Prolonged (18 h) culture with tolbutamide abolished the subsequent KATP channel-dependent and KATP channel-independent actions of tolbutamide and glibenclamide, without altering BRIN-BD11 cell growth, viability, basal insulin release or cellular insulin content (Ball et al. 2000b; McClenaghan et al. 2001).
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Promotion of insulinotropic antidiabetic drug actions by incretin hormones. Experimental diabetes research continues to unravel the intricate mechanisms underlying insulin release, revealing novel cellular targets for the generation of new antidiabetic drugs aimed at correcting ß-cell responsiveness and mass in type 2 diabetes (see McClenaghan & Flatt, 1999b; Bonner-Weir & Weir, 2005). The incretin hormones, GLP-1 and GIP, are potent physiological modulators of glucose-induced insulin secretion, acting through binding and activation of specific ß-cell receptors to promote cAMP and PKA-mediated pathways that regulate insulin exocytosis (Fig. 5). The insulinotropic actions of GLP-1 and GIP have prompted much research interest into their therapeutic potential in type 2 diabetes, either alone, or in combination with other antidiabetic drugs (see reviews by Gault et al. 2003; Green et al. 2004). Given this, and fact that incretin hormones and insulinotropic antidiabetic drugs share key signalling elements, it was of interest to determine whether GLP-1 and GIP could modulate the ß-cell response to different classes of therapeutically relevant insulinotropic agents (Fig. 5).
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Pancreatic ß-cells respond to wide range of nutrient stimuli, which exert diverse cellular effects, including alterations in metabolism and insulin secretion. With a growing body of evidence supporting positive acute actions and negative chronic detrimental effects of different classes of nutrients, it becomes important to gain an appreciation of the contribution of nutrients to physiological maintenance and pathological demise of metabolic and secretory pathways in type 2 diabetes. Cultured BRIN-BD11 cells have proven a particularly useful model for studies of glucose and other hexose sugars (McClenaghan et al. 1996a,d), fatty acids (Dixon et al. 2004), keto acids (McClenaghan & Flatt, 1999c, 2000) and amino acids (McClenaghan et al. 1996b, 1998b; Brennan et al. 2002, 2003; Dixon et al. 2003; Corless et al. 2006). As discussed in the following sections, through provision of a large supply of homogeneous ß-cell tissue, nutrient-responsive BRIN-BD11 cells have offered new possibilities to elucidate and trace complex ß-cell pathways, and the use of advanced research technologies such as 13C-NMR has already yielded novel insights into nutrient regulation of ß-cell metabolic and secretory machinery.
Amino acids and pancreatic ß-cell signalling. Various classes of amino acids are rapidy accumulated within the ß-cell through a number of distinct amino acid transport systems. As illustrated in Fig. 8, as with glucose and fatty acids, amino acids can exert diverse effects on insulin secretion and pancreatic ß-cell function. Three principal modes of insulinotropic action of amino acids have been described in both ß-cells and BRIN-BD11 cells: (a) metabolism and generation of metabolic intermediates including ATP (e.g. leucine), resulting in membrane depolarization; (b) direct depolarization of the plasma membrane through accumulation of positive charge (e.g. arginine); and (c) cotransport with Na+, resulting in membrane depolarization and opening of VDCCs (Fig. 8). Interestingly, many of the amino acid transport proteins are Na+-dependent, and much knowledge regarding the importance of Na+ to the actions of amino acids has come from studies on both isolated ß-cells from the ob/ob mouse (Tengholm et al. 1992; McClenaghan et al. 1997) and clonal rat BRIN-BD11 cells (McClenaghan et al. 1996b, 1998a).
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-aminoisobutyric acid). While the more pronounced effects of alanine (McClenaghan et al. 1997, 1998a) may be attributed to additional ß-cell actions elicited through its efficient metabolism, these data demonstrate that both metabolizable and non-metabolizable amino acids can induce glucose-dependent insulin-secretory responses by modulating electrogenic Na+ transport.
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Effects of metabolic nutrients on expression of ß-cell genes. The most abundant amino acids in the circulation and extracellular fluids are L-glutamine and L-alanine, followed closely by branched-chain amino acids. While glucose and fatty acids are known to regulate insulin secretion and ß-cell integrity through changes in gene expression, the role of amino acids in gene expression had been neglected. These observations prompted recent assessment of the impact of exposure to L-alanine and L-glutamine on BRIN-BD11 gene expression by Affymetrix microarray analysis (Cunningham et al. 2005; Corless et al. 2006). As illustrated in Fig. 10A, prolonged (24 h) exposure to alanine or glutamine, in addition to acutely regulating insulin secretion, demonstrated a positive role of these two amino acids in the regulation of ß-cell gene expression. Notably, exposure to alanine or glutamine upregulated genes related to metabolism, signal transduction, metabolism and oxidative stress, and this was coupled with significant protection against cytokine-induced apoptosis by alanine. Taken together, these observations indicate important long-term effects of amino acids in regulation of function and integrity of pancreatic ß-cells which warrant further investigation.
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-glutamyl cycle (Brennan et al. 2003) and so contribute to ß-cell glutathione production and antioxidant defences. From these studies, it appears that alanine metabolism may influence ß-cell signal transduction and gene expression by generation of key amino acid stimulus–secretion coupling factors (glutamate), generation of lipid metabolites (long-chain acyl-CoAs) and alteration of the cellular redox state (via glutathione generation and related oxidation/reduction). Integrating pathways of nutrient metabolism and insulin secretion. Mitochondria play a central role in cellular energy production and participate in intermediary metabolism, Ca2+ handling and, in ß-cells, glucose-stimulated insulin secretion. As outlined earlier, ß-cells sense blood glucose and other secretagogues and adjust insulin secretion accordingly. Indeed, ß-cell anaplerotic and cataplerotic metabolism may generate coupling factors that contribute to the insulinotropic actions of glucose. While mitochondrial ATP production is essential for glucose-induced insulin secretion, this process also relies on additional signals, including several mitochondria-derived molecules such as citrate, pyruvate and reducing equivalents, and glutamate.
Glutamate is an important metabolic product of glucose, alanine and glutamine metabolism that has gained much attention as a metabolically derived factor that may directly trigger insulin exocytosis (Wiederkehr & Wollheim, 2006). Studies on intracellular glutamate are difficult because glutamate is poorly accumulated into ß-cells, but membrane-permeant glutamate esters provide useful tools to investigate the effects of raised glutamate on insulin secretion. As shown in Fig. 10B, glutamate dimethyl ester (GADME) both initiated insulin secretion at 1.1 mM glucose and potentiated 16.7 mM glucose-induced insulin release from BRIN-BD11 cells, coupled with a rapid rise in [Ca2+]i (Fig. 10C).
Collectively, these data would support the view that glutamate plays an important ß-cell signalling role, and further investigations using membrane-permeant esters should help unravel the exact targets and action of intracellular glutamate and enhance understanding of insulin exocytotic mechanisms. Indeed, glucose, alanine and glutamine represent important physiological ß-cell fuels with key regulatory and intracellular signalling effects which couple metabolism to insulin exocytosis. State-of-the-art NMR approaches provide important new tools with which to study complex metabolic pathways, providing new insights into the relationships between glucose and amino acid handling and insulin secretion.
Chronic detrimental actions of nutrients and the diabetic environment on pancreatic ß-cells
Although ß-cells possesses inherent mechanisms to adapt to nutrient overconsumption to maintain glucose homeostasis, this is balanced by the potentially harmful effects of these same nutrients. Both glucose and fatty acids can exert positive (usually acute) or negative (usually chronic) actions on pancreatic ß-cells, depending on their concentrations and the duration of exposure. Chronic exposure to high glucose concentrations can exert substantive changes to ß-cell nutrient metabolism, and both the hyperglycaemia and hyperlipidaemia of diabetes can alter insulin secretion and ß-cell function. Indeed, the decline in pancreatic ß-cell function in type 2 diabetes has been attributed to so-called glucose toxicity, lipotoxicity and glucolipotoxicity. Moreover, a growing body of evidence supports the view that glucose toxicity and lipotoxicity are interrelated adverse forces on the ß-cell (Prentki et al. 2002). While free fatty acids (FFAs) are physiological ß-cell fuels, chronic exposure to elevated fatty acid concentrations results in a decrease in glucose-induced insulin release, impaired insulin gene expression and an increase in cell death. The exact mechanisms underlying these effects are still under investigation and, although it was initially believed that elevated fatty acid oxidation may decrease glucose oxidation, it now appears more likely that enhanced FFA esterification leading to progressive lipid accumulation may be a major cause of ß-cell failure. This may be coupled with oxidative stress and generation of reactive oxygen species reported under conditions of glucotoxicity and lipotoxicity. While the mechanisms by which high levels of glucose and/or lipids may damage ß-cells have been the subject of intense clinical and experimental investigation, less attention has been directed to other diet-derived factors, including the amino acids.
Impact of homocysteine on pancreatic ß-cell function. As outlined above, in type 2 diabetes ß-cell dysfunction may arise following chronic increased exposure to both glucose and fatty acids. Obesity and type 2 diabetes are diseases of the metabolic (insulin resistance) syndrome, a cluster of metabolic abnormalities encompassing hyperlipidaemia and cardiovascular disorders. Elevated circulating homocysteine and hyperhomocysteinaemia has emerged as important risk factors for cardiovascular disease and other diseases of the metabolic syndrome, including type 2 diabetes. However, while raised plasma homocysteine has been reported in obese subjects with defective insulin secretion and insulin resistance, and homocysteine has been demonstrated to exert adverse effects on neural and vascular cells, perhaps surprisingly the effects of homocysteine on pancreatic ß-cell integrity and secretory function have not yet received due attention.
Acute exposure of BRIN-BD11 cells to homocysteine induced a concentration-dependent decline in both basal and glucose-induced insulin secretion, and has also been demonstrated to exert a generalized impairment of ß-cell function (Patterson et al. 2006). The detrimental action of homocysteine on glucose-induced insulin secretion was coupled with a reduction in TCA cycle-dependent glucose metabolism, with decreased labelling of glutamate from glucose at positions C2, C3 and C4. However, these acute effects of homocysteine could not simply be attributed to adverse actions on cellular insulin content, cell viability or apoptosis/necrosis (Patterson et al. 2006). Thus, these data indicate that homocysteine impairs insulin secretion through alterations in ß-cell glucose metabolism and generation of key stimulus–secretion coupling factors.
This being so, the participation of homocysteine in the demise of the pancreatic ß-cell in diabetes warrants further investigation. Such studies should also consider the effects of chronic ß-cell exposure to homocysteine and aim to dissect the exact nature of the homocysteine-induced effects, including assessment of the contribution of homocysteine-derived hydrogen peroxide to the detrimental actions of this amino thiol. Future investigations should also consider the effects of homocysteine on pancreatic islet function in vivo together with associated metabolic effects. However, as previously noted with glucose and fatty acids, the current data represent compelling evidence for the existence of ß-cell amino acid desensitization/toxicity, consistent with the view that overnutrition can result in adverse ß-cell events that contribute to the pathogenesis of diabetes.
Concluding remarks
Sir Edward Sharpey-Schafer adopted a pioneering and persistent approach with passion and drive, providing a framework for ‘New (chemical) Physiology’. Despite ‘years of drought’, he followed his belief in specific discoverable substances that produced profound effects on the human body, demanding that endocrinology be a legitimate and respectable experimental science, paving the way for the discovery of hormones. As such, Sir Edward demonstrated that experimental physiology could provide a firm basis for medical practice, an important message for every aspiring research scientist. This review outlines key aspects of my research to date, demonstrating that innovative bioengineering approaches may provide new and much needed sources of functional ß-cell tissue for experimentation and potential therapy. Data derived from experimentation on bioengineered ß-cells, intact and dissociated islets have greatly enhanced knowledge of diverse and complex attributes of the ß-cell. Such studies have directly contributed to therapeutic advances, offering new approaches to diabetes control. These include new classes of insulinotropic agents targeting KATP channels and other elements regulating ß-cell signalling, and other agents, such as GLP-1 and GIP, that can influence ß-cell proliferation, survival, neogenesis and function, representing the next generation of antidiabetic drugs. This review highlights how bioengineered ß-cells can provide important new insights into cell signalling and function, offering exciting new research possibilities, including the establishment of sites and actions of insulinotropic agents, the understanding of cell desensitization and demise in diabetes, and the unravelling of complex metabolic and insulin-secretory pathways. As illustrated in Fig. 11, there are numerous possible ß-cell targets for insulin-releasing drugs and, as such, experimental ß-cell physiology will play an important part in the discovery and characterization of novel and improved therapeutics for the emerging diabetes epidemic.
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Footnotes
This lecture was given at the Main meeting of the Physiological Society at University College London on Wednesday 5th July 2006.
References
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Acknowledgements
It is a great personal honour to have received this award and, accordingly, I wish to express my gratitude to the Sharpey-Schafer family for generously supporting this triennial Lecture and Prize and the Members of The Physiological Society Prizes & Prize Lectures Sub-Committee and Society Council who elected me to be the recipient of the prestigious Sharpey-Schafer Lecture & Prize 2005.
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