2017 Group 3 Project
Note: Throughout this page, terms marked with an Astericks (*) will be found in the glossary.
- 1 Introduction
- 2 Structure
- 3 Pancreatic and Beta Cell Development
- 4 Function
- 5 Signalling
- 6 Role in Pathology
- 7 Current Research
- 8 Review Quiz
- 9 Glossary
- 10 References
Beta cells, abbreviated as β-cells, are one of the four major cells found within the endocrine* portion of the human pancreas*. The human pancreas is a composite organ, which extends from the distal foregut endoderm and is composed of an exocrine* and endocrine portion. The exocrine tissue consists of copious amounts of acinar cells, that discharge digestive fluid, and a complementary duct complex through which the secreted fluid drains into the intestine. The endocrine portion of the pancreas is composed of islets of Langerhans*, or pancreatic islets (reviewed in). These islets of Langerhans constitute approximately 1-2% of the pancreas and are dispersed throughout it. They contain multiple endocrine cells; alpha, beta, delta, gamma and epsilon cells, which secrete the hormones; glucagon, insulin, somatostatin, pancreatic polypeptides and ghrelin, respectively. The pancreatic islets also host several endothelial cells, nerves and fibroblasts hence deeming them highly vascularised structures, which vary in size from small clusters to larger clusters, containing up to several thousand islets and reaching diameters of 300-400 μm. As the majority of pancreatic islets are typically less than 160 μm in diameter, islets which exceed this diameter are considered to be responsible for the majority of islet's total mass.
Currently, it is predicted that adult humans have approximately 2 million islets of Langerhans, which contributes to 2% of their total pancreatic weight. Within these islets, approximately 60-70% of the total islet-cell population can be attributed to beta cells. Beta cells, or insulin-secreting cells, are thus key cells within the pancreas and are predominately responsible for preserving normoglycaemia* by neutralizing blood glucose levels with appropriate quantities of insulin secretion. While glucose levels are the chief driver, insulin secretion and synthesis by beta cells may occasionally occur in response to other factors such as nervous stimuli, hormones and nutrients.
|Video 1. Introductory Video on the Role of the Pancreas:|
|Table 1. History of Pancreatic and Beta Cell Development|
|1869||The islets of Langerhans, or pancreatic islets, were first discovered by German anatomist Paul Langerhans, within rabbits, as anatomical formations with no direct connection to the duct. At that moment, no function was attributed to them. Prior to the discovery, the pancreas was solely recognised as an exocrine organ.|
|1889||Joseph Von Mering and Oskar Minkowski successfully completed the first pancreatectomy* in a dog. Through this, they discovered that the removal of the pancreas induced diabetes in the dog . This discovery was paramount as it irrevocably established that the pancreas and diabetes were affiliated .|
|1893||Edouard Laguesse, who located islets in the human pancreas, suggested that they were responsible for the internal secretions that regulate glycemia*, and named them Langerhans, after their discoverer .|
|1907 and 1911||Lane and Bensley developed staining techniques to analyse the structure of the pancreas at a cellular level. These stains revealed at least two different granular cell types; A cells, which contained granules preserved by alcohol, and B cells, which contained granules preserved by chrome sublimate*.|
|1914||Homans noticed that B-cells were involved in experimental diabetes and thus attributed the sugar regulating function to them.|
|1921||Best and Banting isolated insulin from the canine pancreas after ductal ligation. Their discovery of insulin was awarded the Nobel Prize in Medicine in 1923.|
|1943||Dunn et al. selectively destroyed beta cells by administration of alloxan*, which led to diabetes.|
|1957-1974||Morphological features of the islet cell types were established through multiple studies utilising electron microscopy. Their structure and the appearance of their cytoplasmic secretory granules were observed.|
|1966||First case of insulin independence by pancreas transplantation by William Kelly and Richard Lillehei into a 28 year old uremic* woman at the University of Minnesota.|
|1972||Pictet and Rutter analysed the ultrastructural development of the pancreas by transmission electron microscopy and found that both the endocrine and exocrine cells of the pancreas were derived from a common progenitor*; alpha cells were the first endocrine cells to differentiate.|
|1977||Cudworth et al. made a crucial step in the etiological* classification of diabetes; introducing the terms type 1 and type 2 diabetes.|
|1990||Sharp et al. led further advances in transplantation achieving clinical insulin independence, lasting 1 month, through islet transplantation.|
|2000||Shapiro et al. utilise the "Edmonton protocol*" where 7 patients with type 1 diabetes receive islets and all recipients achieve insulin independence. The study utilised immunosuppression drugs to prevent rejection.|
|2014||Pagliuca et al. conduct the first successful generation of functional human pancreatic beta cells in vitro*, potentially providing an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes .|
|Present||Research continues into stem cell therapeutics for diabetes (refer to "Current Research").|
Among most species, pancreatic islets are generally characterised by an oval shape although the distribution of cells, within the endocrine pancreas, are subject to variation across animals. In most mammals, the islet contains a core of beta cells, which is encircled by a mantle of non-beta cells. Conversely, in humans and primates, cells are dispersed with greater complexity and deviate from the mammalian mantle-core arrangement to a composite of several mantle-core subunits. Due to such cellular organisation, pancreatic islets within humans and primates may be of oval or clover-leaf shape. Islets of Langerhans are highly vascularised structures, which contain capillaries that are lined by endothelial cells comprising of mitochondria*, ergastoplams*, a golgi complex* and a nucleus*. The peripheral extensions of the cytoplasm* of such endothelial cells are typically thinner and lack these organelles however, they contain several fenestrae or openings. While it is not confirmed whether these fenestrae vary in quantity and/or distribution, it is predicted that they represent areas for communication and allow the transfer of materials between the bloodstream and the extravascular space.
The beta cell, itself, is enclosed by a continuous plasma membrane and includes, within its cytoplasm, several membrane-bound organelles such as the endoplasmic reticulum*, mitochondria, a nucleus, golgi complex and secretory granules*. The endoplasmic reticulum, also called the ergastoplasm, is composed of two lamellar membranes that have ribosomes attached to their exterior surface. In comparison to the mitochondria present within adjacent acinar cells, the mitochondria of the beta cells are relatively smaller in size. However they are easily identifiable by the cristae within their matrix. The golgi complex and beta granules are similarly composed of smooth membranous sacs, although the latter is sequestered from the cytoplasm by the walls of these sacs. The beta granules are additionally dispersed throughout the entire cytoplasm. The nucleus contains a double-layered nuclear membrane and also several nuclear pores, which allow continuity and interaction between the nucleoplasm and cytoplasm of the beta cell.
Adjacent beta cells are connected to each other at distributed focal points, termed desmosomes, along their plasma membrane. Between two successive desmosomes, an intercellular space exists and its size is reliant upon the respective activity of the cells. The capillaries present within the islets of Langerhans are detached from the beta cells by two basement membranes; one related to the endothelium and the other to the capillary surface. Typically, between these two basement membranes, cross-sections of fibroblasts* and unmyelinated* nerve fibres can be detected. In some instances, unmyelinated nerve fibres have also been identified beneath the basement membrane and in close contact with the cell’s plasma membrane. Although presently unconfirmed, this is considered to indicate synapses between the nerve fibres and the beta cell.
Structural Modifications with Age
Extensive studies conducted on human pancreases, from the prenatal period through to adulthood, have revealed that the anatomical architecture of pancreatic islets resemble their adult form by the age of 2. As a direct result of this, the pancreas experiences substantial beta cell proliferation* during the first 2 years after its conception, which enables significant increases in beta cell mass and area. Increased beta cell production thus manifests as an increase in the diameter of existing pancreatic islets, rather than the formation of new islets. In fact, while 10% of beta cell mass increase during early life can be attributed to islet development, a sizeable 90% can be linked to an increase in islet size. Although, beta cell replication continues throughout life, the replication rate notably decreases after adolescence and hereafter beta cell mass remains relatively constant between the ages of 20 – 100 (reviewed in).
Structural Comparison between Humans and Mice
Consequential of the clinical importance of pancreas-associated disease (refer to "Pathology"), the structure of the pancreas is studied extensively within medical science. Studies of the human pancreas, nevertheless, are restricted due to the inherent ethical and medical limitations present in obtaining pancreatic samples at specific and intermittent intervals. As a result of scarce human pancreatic samples, animal models are increasingly used in scientific studies. Within current pancreatic research, mice are the most frequently studied animal models. As a sizeable portion of scientific knowledge concerning the human pancreas is derived from studies conducted on mice, it is thereby imperative to identify and understand the distinction between the human and mouse pancreas.
At a macroscopic level, the human pancreas is a well-defined organ, which contains three key structural components: the anterior and posterior head, body and tail (reviewed in). The left border of the superior mesenteric artery borders the head and body of the pancreas, while the midpoint of the pancreatic body and tail, combined, is understood as the border between the body and the tail. Reflecting a C-shape, the pancreatic head is continuous with the upper curvature of duodenum whereas the pancreatic tail touches the hilum of the spleen. The narrow and flat pancreatic body locates itself underneath the stomach and extends in such a manner that it crosses with the superior mesenteric artery and vein, abdominal aorta, inferior vena cava and portal vein.
Contradictorily, the mouse pancreas is less defined than its human counterpart, see Figure 2. and Figure 3., and in a dendritic manner, is uniquely diffused along the mesentery of the proximal small intestine of mice. Analogous to the human pancreas, it can also be distinguished into three key structural components. However, these are termed ‘lobes’ and are identified as the duodenal, splenic and gastric lobe. The largest lobe, the splenic lobe, extends between the duodenum and the spleen and is homologous to the body and the tail of the human pancreas. The second largest lobe, the duodenal lobe, is located within the mesentery, that surrounds the duodenum, and is homologous to the human pancreatic head. Additionally, the gastric lobe, which is the smallest observable lobe, is considered to be a component of the splenic lobe, from which it originally develops, and is homologous to the auricle of the human pancreas. Characteristically, patches of adipose*, connective and lymphatic tissue separate these three main pancreatic lobes in mice.
Microscopically analysed, each lobe of the pancreas contains numerous smaller lobules, which vary in size respective to the organism and measure 1-10 mm in diameter in humans and 0.5-1.5 mm in diameter in mice. Dome-resembling clusters of pyramidal acinar cells are dispersed throughout these pancreatic lobules. They secrete various digestive enzymes, which are ultimately released into the duodenum by successive movement through intercalated ducts, intralobular ducts, interlobar ducts and finally the pancreatic duct, thus forming the exocrine pancreas. The endocrine portion of the pancreas, predominately constituting islets of Langerhans, is embedded within the exocrine tissue. Notably, the size and distribution of pancreatic islets is congruous between humans and mice. With distinctly irregular cross-sections, they are home to thousands of hormone-secreting endocrine cells, which may also be singularly and randomly scattered across acinar and ductal tissues. While a minimum of five different polypeptide endocrine cells can be identified within the islets, beta cells, which synthesize and secrete insulin, form the majority and account for 60-70% of the islet cell population in humans and 60-80% in mice.
The spatial organization of beta cells has continuous focus for scientific research. In mice, the mantle component of pancreatic islets contain non-beta cells while the core component exclusively comprises beta cells. In humans, it was initially perceived that, alongside a similar mantle-core pattern, beta cells additionally formed ribbon-like clusters and were scattered throughout islets in an unorganised manner. However, consequential of recent studies, the current scientific consensus is that human pancreatic islets form trilmainar plates, which are folded into a U- or O-shape, and within which there are two layers of alpha cells that enclose a single beta cell layer. The reason for this specific organisation is considered to be the promotion of heterologous contact between both endocrine cells. Alternatively, in mice, there is homologous contact among alpha cells within the mantle of the islet and beta cells within the core of the islet. It is proposed that this specific type of spatial arrangement promotes alpha-cell-stimulation of beta cell functional activity.
|Table 2. Summary Table of Key Comparisons between Human and Mice Pancreas|
|Macroscopic Pancreatic Structure||Well-defined organ with three structural components: the head, body and tail||Less-defined structure with three lobes: duodenal, splenic and gastric lobe|
|Diameter of Lobules||1 - 10 mm||0.5 - 1.5 mm|
|Beta Cells as a % of Pancreatic Cell Population||60 - 70%||60 - 80%|
|Microarchitecture of Islets||Trilaminar islets predominate||Mantle islets predominate|
|Contact between Alpha and Beta Cells||Heterologous contact between endocrine cells as two layers of alpha cells enclose a single beta cell layer||homologous contact among alpha cells within the mantle of the islet and beta cells within the core of the islet|
Pancreatic and Beta Cell Development
The ultimate architecture of the complete adult pancreas is the outcome of several necessary embryologic events (reviewed in). During embryonic days 8.5 and 9, the emerging embryo moves from a lordotic position to a fetal position, which causes the endoderm layer to fold on itself and form a primitive gut tube that is divided into the foregut, midgut and hind-gut. Upon the establishment of the gut tube, the development of the dorsal pancreas is hereafter controlled by the notochord, which is the source of permissive signals that enable the differentiation of the foregut endoderm into a pancreas. The removal of the notochord is scientifically evidenced to eliminate the expression of pancreatic genes thus inhibiting pancreatic development and preventing the formation of all pancreatic cells, including beta cells).
Of the three divisions of the gut tube, the foregut is the most essential as the first morphological process in pancreatic formation, and beta cell development, is a condensation of the mesenchyme covering the dorsal aspect of the endodermal gut tube in the foregut. Succeeding this condensation, at 26 days post-conception, the endoderm evaginates into the mesenchyme and dorsal and ventral buds arise from the foregut. The buds are marked by transcription factors, such as pancreatic and duodenal homeobox factor 1 (PDX1) , SRY (sex-determining region Y)-box 9 (SOX9) and GATA binding protein 4 (GATA4), which are all necessary for pancreatic growth. The epithelium, which constructs the mesenchyme surrounding the pancreas, coelomic epithelium, also begins to compartmentalise the pancreas away from non-gut structures and a substantial increase in the proliferation of mesenchymal cells, between the coelomic epithelium and pancreatic epithelium, is seen.
At approximately 37 to 42 days after gestation, the proximal portion of the dorsal bud form small accessory ducts called ducts of Santorini, while the remainder of the dorsal bud and ventral bud fuse to form the duct of Wirsung, which runs across the entire pancreas. Hereafter, the cellular architecture of the pancreas undergoes significant change and a major amplification in the amount of endocrine cells, particularly β-cells, is observed. Pancreatic acinar cells also undergo cell differentiation and gene expression for acinar enzymes increase in an exponential manner. The rough endoplasmic reticulum and zymogen granules also begin forming, consequently causing the pancreas to become opaque to the naked-eye due to high zymogen granule concentrations.
Although pancreatic alpha cells are differentiated and observed before beta cells, the earliest endocrine cells of the pancreas are collectively found in an admixture of endocrine, exocrine and epithelial cells, which all maintain close contact with the lumen. Eventual morphological transitions, however, allow endocrine cells to become distinct from epithelial cells and lose connection with the lumen. At this stage in development, a down regulation of PDX1, which marks pluripotent precursor cells, is detected in endocrine progenitor cells as they become non-epithelial. Throughout pancreatic maturation, PDX1 remains principally expressed in the beta cells of the pancreas population, which is considered to be necessary for proper endocrine differentiation. As the developmental process nears conclusion, endocrine cells aggregate in pancreatic ducts, as shown in Figure 4., and form islets of Langerhans.
On a hormonal level, at around 52 days after the conception of the human pancreas, Neurogenin-3 (NGN3), a regulator of pancreatic islet differentiation and regeneration is first detected in the pancreas. Although the expression of the transcription factor PDX1 precedes them, insulin, glucagon and somatostatin are the first pancreatic hormones expressed. Pancreatic polypeptide is not detected until about 10 weeks post-conception. At around 12 weeks into the gestation period, at the point where pancreatic islet formation reaches its peak, all four hormones are expressed and thus the total number of hormone expressing cells, present within the islets, is increased.
The overall function of the pancreas is to maintain glucose levels within the blood and beta cells play a specialised role within this process. The insulin gene, which provides instructions for the manufacture of insulin, is solely expressed in the endocrine portion of the pancreas within the beta cells of pancreatic islets. Insulin, a polypeptide hormone, is a key regulator of metabolism as it promotes the storage of excess glucose, amino acids and fatty acids by acting on the liver, muscle and adipose tissue. Insulin release into the bloodstream predominantly occurs as a consequence of increased glucose levels detected in the blood. Glucose phosphorylation, by glucokinase, is currently understood to act as the glucose sensor as it adjusts the rate of cellular metabolic activity to the extracellular concentration of glucose. Glucose metabolism generates intracellular signals in beta cells, which subsequently initiates insulin secretion, insulin mRNA translation and insulin gene transcription.
|Video 2. Video Elucidating the Function of Insulin in the Human Body:|
Insulin Gene Expression
In the late 1980s, the 5’ flanking region of the insulin gene was identified in transgenic mice. This proved to be an extraordinary breakthrough in science as it allowed the general location of binding sites, for transcription factors, to be discovered. The transcription factor binding sites, which direct beta-cell-specific expression, were eventually located to be between −520 and +1 base pairs relative to the transcription initiation site. In later studies, congruous expression data was collected for the human insulin gene. Both results thus collectively indicate that there are sequences conserved between mammalian insulin genes, which lie within 350bp of the transcription start site and control the specific cell-type expressions. The enhancer region in mammalian insulin genes is located between nucleotides -340 and -91. While the transcription factors that bind onto this region principally determine the glucose-regulated expression properties of the gene, the activity of the enhancer can be additionally up-regulated and/or down-regulated by cellular activity. For example, through autocrine signalling, the insulin secreted from beta cells may increase insulin transcription in the same beta cells, by positively influencing enhancer-mediated activation.
In the 1950s, insulin, as show in Figure. 5, became the first protein to have its structure clearly revealed. The discovery exposed insulin as a two-chain hormone and consequently triggered widespread speculation in the scientific community on the underlying mechanisms contributing to such a structure. Succeeding the discovery of single-chain precursor proteins, which occurred in 1967, insulin synthesis was finally understood to occur through several reactions with multiple single-chained precursor proteins, including preproinsulin and proinsulin.
During insulin biosynthesis, the initial product formed instantaneously after insulin mRNA translation is preproinsulin, which contains a hydrophobic N-terminal 24 residue signal peptide. The signal peptide engages with the signal recognition particle (SRP), a ribonucleoprotein particle that is located within the cytosol, which enables the separation of the preproinsulin polypeptide chain from the cytosolic compartment into the secretory pathway. From here, the preprohormone is translocated across the membrane of the rough endoplasmic reticulum (RER) and into its lumen, via a peptide-conducting channel. A specialized signal peptidase, situated on the surface of the RER membrane, cleaves and rapidly degrades the signal sequence. Inside the RER, several chaperone proteins catalyse the folding of proinsulin and facilitate its manufacture of three disulfide bonds, which largely contribute to the development of its native structure.
Within a time period of ~10-20 minutes, proinsulin is rapidly transported from the RER to the Golgi apparatus. Here, proinsulin is packaged into immature secretory vesicles and cleaved to form equimolar amounts of insulin and connecting polypeptide (C-peptide) . The predominant route for proinsulin processing is via cleavage at the junctions resulting in a 32,33 proinsulin peptide split, followed by the removal of 2 amino acids resulting in Des-31,32 proinsulin which finally forms mature C-peptide and insulin by the action of pro-protein convertase-2 (PC2) and carboxypeptide-E (CPE), see Figure. 5. The alternative pathway, which occurs less commonly, involves the removal of 2 amino acids are first. The formation of insulin from its precursor, proinsulin, is initiated approximately 20 minutes after the synthesis of preproinsulin and typically lasts for 1-2 hours, independent of additional protein synthesis.
While proinsulin and insulin have a direct biological effect, C-peptide has no effect on homologous or heterologous tissue and no ability to influence the concentration or action of beta cell hormones (reviewed in). Although small volumes of proinsulin and its immediate cleavage forms, C-peptide and insulin, are collectively stored in the secretion granules of beta cells, insulin is the predominant hormone released after signalling. Notably, proinsulin and insulin share similarities with respect to many of their properties, including solubility, isoelectric point, self-associative properties and relative reactivity with insulin antiserum. The insulin moiety found within proinsulin is also highly similar to the one present in insulin itself, thus making their overall conformation highly similar.
Cell Biology of Insulin Biosynthesis
Congruous to other neurosecretory cells, the Golgi apparatus plays a substantial role in the formation of secretory granules in beta cells. However, its dynamic nature and the underlying mechanisms, which facilitate the transport and sorting of secretory products within it, is currently only partially understood. With further research, new models for Golgi progression are continuously proposed. Currently, a scientifically favoured model presents a form of the Golgi apparatus wherein individual cisternae, containing their secretory contents, migrate from cis to trans while small, coated vesicles transport various Golgi resident proteins and enzymes in an opposite manner from trans to cis. Relative to this model, while secretory products mature, they are kept in the same compartment and only exit from it as they dissipate into the trans Golgi network into immature secretory granules. Recent immunocyto-chemical studies have established that newly synthesized clathrin-clad granules in the trans Golgi cisternal network (TGN) are rich in proinsulin, which confirms the proposal that proinsulin conversion to insulin occurs principally during the maturation of the secretory granules. The proteolytic processing of proinsulin may be prevented by energy poisons, which block their transfer into secretory vesicles. Nevertheless, once newly manufactured proinsulin has reached the trans Golgi, energy is no longer required to convert it to insulin. While proinsulin conversion may begin in the trans compartment of the Golgi apparatus, it is now confirmed that it occurs predominantly within newly manufactured secretory vesicles as they leave the Golgi region and mature biochemically in the cytosol.
Quantitative Aspects of Insulin Secretion
In the late 1960s, the dynamics of insulin secretion, in response to glucose stimulation was studied and results demonstrated that glucose infused into the pancreas for extended amount of time, led to two different phases of insulin release. The first phase was characterised by rapid insulin release for 2 minutes, while the latter phase involved the continuous release of insulin, which slowly increased in rate until the termination of the glucose stimulus.
This was the first indication that insulin release from beta cells follows a biphasic pattern. Insulin release in the initial phase is due to ATP-dependent potassium channels, which allow increased calcium influx and the discharge of ready and releasable insulin-containing granules. The second phase involves preparing granules for release and is thought to include the translocation of granules and checks to ensure that they are competent for exocytosis. Insulin secretion in the second phase therefore occurs over a greater length of time. In fact, in mouse islets, of the 13 000 granules present in beta cells, only ~100 are ready for release in phase one and thus the others must be finalized in phase two. Quantitative comparisons can be made between both phases of insulin release by reports, which state that first and second phase insulin secretion in mouse islets occur at a rate of 0.14%/min and 0.05%/min, respectively.
The Stimulus-Secretion Coupling Mechanism
Stimulus-secretion coupling refers to glucose-dependent insulin secretion from beta cells. Congruous to muscle cells, beta cells are also dependent on electrical activity and calcium entry in order to discharge insulin into the body. As a result, they have specific ion channels located in their plasma membrane, which enable the flow of calcium and potassium ions into and out of the cell. As these ions are electrically charged, their movement across the membrane causes action potentials, which are sharp changes in voltage.
In pancreatic beta cells, an increase in blood glucose levels block ATP-dependent potassium channels thus leading to the depolarisation of the membrane. Initially identified in ventricular myocytes, ATP-sensitive potassium channels are potassium channels that are inhibited by high intracellular concentrations of ATP (reviewed in. As the activation of these channels disallow the production of action potentials, their inhibition, by glucose, stimulates action potentials by triggering voltage-dependent calcium channels in the membrane to open and thus allow increased calcium influx into the cell (reviewed in). Within the cell, the calcium ions act on exocytotic machinery to stimulate the merging of vesicles, containing insulin, with the plasma membrane, thereby preparing them for secretion into the blood stream .
Conversely, as the electrical activity induced in beta cells is mainly due to calcium ion influx , decreased extracellular levels of calcium ions prevent the firing of action potentials and thus, by extension, inhibit insulin secretion .
Glucose Metabolism and Potassium Channels
As glucose breakdown is imperative for insulin secretion, the inhibition of mitochondrial metabolism prevents insulin from being secreted by beta cells. Adenosine triphosphate (ATP) is the major product of glucose metabolism and therefore is a key factor that links the mitochondrial metabolism of glucose and ATP-sensitive potassium channels. ATP-dependent potassium channels contain four pore-forming subunits alongside four accessory sulfonylurea receptor subunits (SUR1). These SUR1 subunits are essential components of beta cells, as they are the focus site for anti-diabetic sulphonylurea drugs, which aim to increase insulin secretion by replicating the effect of glucose in order to block ATP-sensitive potassium channels.
While increased glucose levels generate a high cytoplasmic concentration of ATP and depolarize beta cells by blocking ATP-sensitive potassium channels, under low concentrations of glucose, these channels are open and enable the outward flux of potassium, causing a negative cellular membrane potential of ~ -70 mV. At this point, action potentials cease to occur and insulin exocytosis is inhibited. The membrane potential is only increased to more positive values once glucose-induced increases in ATP drive the closure of ATP-sensitive potassium channels. Hereafter, L-type voltage-dependent calcium channels, which are predominately expressed in beta cells, allow the release of insulin into the blood in order to maintain glucose homeostasis.
Un-Coupling Glucose Metabolism and ATP Production in Beta Cells
As mentioned above, the voltage of the cell membrane is dependent upon changes in the intracellular concentration of ATP. As a result of this, perturbations of metabolic pathways that produce ATP can, by extension, impact insulin secretion by beta cells. In all cells, ATP is generated in the mitochondria, which produces approximately 95% of the cell’s energy and is therefore colloquially termed “(the) powerhouse of the cell”.
Within the mitochondria, the electron transport chain (ETC), as depicted in Figure 7., is the central machinery responsible for ATP production (reviewed in) and is heavily reliant upon a proton gradient across the mitochondrial membrane. The four enzyme complexes of the ETC transfer electrons from donors, such as NADH, to the ultimate electron accepter, oxygen. Throughout this transfer of electron, the ETC concurrently pumps protons, or hydrogen ions (H+), into the inter-membrane space, which subsequently generates a gradient across the inner mitochondrial membrane. ATPase, an enzyme, then harnesses this gradient to drive ATP synthesis. This process is additionally explained in Video 3.
Specifically within beta cells, the uncoupling protein-2 (UCP2) can largely impair the generation of ATP via the mitochondrial ETC as it enables a proton leakage across the mitochondrial membrane and therefore inhibits the necessary formation of a proton gradient. The over-expression of UCP2 can therefore bypass the production of ATP, which is reinforced by studies that report a 50% reduction in ATP content in the cells of UCP-2-induced pancreatic islets). On the other hand, a loss of UCP2 expression is hence responsible for increased intracellular ATP concentrations and glucose-stimulated insulin secretion by beta cells, which serves to decrease levels of glycemia). It has also been discovered that the expression of Sirt1, in beta cells, downregulates UCP2 expression, further enhancing insulin secretion .
|Video 3. Explanation of the Mitochondrial Electron Transport Chain:|
Role in Pathology
As established above, insulin is vital in the maintenance of glucose levels in blood. Through the signalling of liver, muscle and fat cells, insulin enables glucose uptake and allows for its use as an energy source. When the body has a sufficient amount of energy, insulin signals the liver to take up excess glucose and store it as glycogen. Thus, in the absence of insulin or responsiveness to insulin, glucose remains in the blood stream resulting in a rise of blood-glucose levels, leading to diabetes. This concept is reiterated visually in Video 4.
|Video 4. Explanation of the relationship between insulin, glucose and diabetes:|
The role of beta cells in disease in explained in Table 3.
|Table 3. Comparison of Type 1 and Type 2 Diabetes Mellitus|
|Type 1 Diabetes Mellitus (T1DM)||Type 2 Diabetes Mellitus (T2DM)|
Type 1 diabetes is one of the most common chronic childhood diseases:
Type 1 diabetes varies dramatically on a global scale with >350-fold variation in incidence among reporting countries; overall:
Type 1 diabetes results from an insulin deficiency that is caused by the loss of pancreatic beta cells. This can be consequential of:
Early familial and twin studies support the importance of both genetic and environmental risk factors:
Primarily lifestyle factors cause this insulin resistance to occur. Such factors may include:
The pathogenesis of autoimmune-induced type 1 diabetes has been extensively studied through the use of animal models, such as non-obese diabetic mice and diabetes-prone BioBreeding rats. From the studies, exact mechanisms of initiation and progression remain unclear, however through the it is generally believed that beta-cell auto-antigens, macrophages, dendritic cells, B lymphocytes and T lymphocytes are involved in the beta-cell specific autoimmune process. As seen in Figure 8., the beta-cell auto-antigens are processed by the macrophages, dendritic cells or B cells in the pancreatic islets and then presented to the auto-reactive CD4+ T cells in the peripheral lymphoid system. These cells are consequently activated, thus leading to the secretion of cytokines, which may activate beta-cell specific cytotoxic CD8+ T cells. Activated T cells also recruit at the islets, producing cytokines which further activate macrophages and other T cells; all contributing to the destruction of pancreatic beta-cells.
Pancreatogenic (Type 3c) Diabetes
Unlike T1DM and T2DM, Type 3c Diabetes Mellitus (T3cDM) is a form of secondary mellitus that is caused by underlying pancreatic diseases such as acute or chronic pancreatitis, cystic fibrosis, pancreatic cancer or other form of pancreatic trauma leading to loss of pancreatic tissue; the most common cause being chronic pancreatitis (CP). CP accounts for 75-80% of T3cDM cases, while pancreatic carncinoma accounts for 8%. T3cDM was initially believed to have a lower prevalence (approximately 0.5-1.2%) among diabetes cases in North America, however recent studies suggest higher occurrences than originally anticipated. In fact, a recent study shows a 5-10% prevalence among all diabetic subjects in Western populations. A review by Anderson et al. (2013) suggests that previous underestimations may be attributed to the fact that it has become easier to detect exocrine pancreatic pathology, and quantify exocrine dysfunction, with new advancements in imaging technology and noninvasive screening methods.
Moreover, the endocrinopathy is far more complex in T3cDM, than T1DM and T2DM, due to additional comorbidities. As a result of the cross over in risk factors for T2DM and T3cDM, such as obesity and congruous symptoms associated with all three forms of diabetes; e.g. difficulties with glucose homeostasis, a method of discrimination between the diseases is required. The proposed criteria for this involves noting
- Presence of exocrine pancreatic insufficiency (monoclonal fecal elastase-1 test)
- Pathological pancreatic imaging (endoscopic ultrasound, MRI, CT)
- Absence of type 1 diabetes mellitus associated autoimmune markers
- Impaired beta cell function
- No excessive insulin resistance
- Impaired secretion of incretins
- Low serum levels of lipid soluable vitamuns
However, despite the implementation of the proposed criteria, overlapping still occurs as patients suffering from T1DM and T2DM, for an extended period of time, are also victims of pancreatic failure and are at risk of acute and chronic pancreatitis. Additionally, patients with pancreatitis may develop T1DM or T2DM independently. Thus, the best discriminator between all three forms of diabetes is believed to be the pancreatic polypeptide's (PP) response to nutrients .
Interestingly, studies have revealed that isolated hepatic insulin resistance coexists with clinical studies of T3cDM . This is documented to be consequential of CP, pancreatic cancer and/or cystic fibrosis, which results in unsuppressed hepatic glucose production. PP deficiency was also found to coincide with T3cDM and thus, is identified as a likely mediator of the hepatic defect. Animal studies indeed confirm that PP deficiency results in diminished hepatic insulin receptor availability and thus PP administration reverses hepatic insulin resistance in patients with CP, thereby improving glucose tolerance.
How cancer induces T3cDM:
The most likely explanation for the consequential occurence of diabetes in pancreactic ductal adenocarcinoma (PDAC) patients, is a paraneoplastic phenomenon caused by tumor-secreted products. One such product that results in beta-cell dysfunction is adrenomedullin; a pluripotent hormone with homology to amylin, which is over-expressed in PDAC. Adrenomedullin receptors are found on beta cells and therefore external administration of adrenomedulin causes the inhibition of insulin secretion, as evidenced in animal studies One such study performed by identified adreninedulin (AM) to impair insulin secretion from beta cell and cintributing to the insulin inhibitory effect of pancreatic cancer cells in vitro and in vivo* (as seen in Figure 10.). These finding support that adrenomedulin is mediator of beta cell dysfunction in PDAC.
How chronic pancreatitis induces T3cDM
Development of diabetes in chronic pancreatitis patients mainly occurs due to the destruction of islet cells by pancreatic inflammation. Additionally maldigestion caused by pancreatitis leads to diminished insulin release. GLP-1 is an incretin hormone secreted by the L-cells of the distal intestine, and has the effect of stimulating insulin gene expression and proinsulin biosynthesis. GLP-1 also has proliferative and anti-apoptotic effects on the beta-cell. Thus, maldigestion leads to impaired incretin secretion and therefore diminished insulin release of the remaining beta-cells. In contrast to the autoimmune-mediated destruction of beta-cells in T1DM, gulcagon-secreting alpha cells and PP cells are also subject to destruction in CP leading to a complex metabolic situation.
Insulin injections are used in place of the insulin that would normally be produced by the body. Individualised treatment plans are suggested for patients however, generally T1DM patients take 3-4 injections per day to provide optimal glucose control, while T2DM take 2 injections per day if not in conjunction with other medication. Insulin shots are most effective when administered when food enters the blood, such as 30 minutes before eating. Furthermore, particular sites for injections, such as the abdomen, provide better results while insulin administered at the arms and thighs take a longer period of time to enter into the blood stream.
However, insulin vials and syringes provide an inconvenience to patients. Adverse psychological and social effects may occur to others, and to the patient, when using syringes. Incorrect insulin use and dosage could also result in risks. Insulin pens have thus been developed to counteract these issues by providing rapid, long-acting insulin. Nevertheless, insulin pens have a greater cost per unit of insulin compared to the insulin supplied in vials.
Insulin pumps are another method of attaining insulin and counteracting insulin deficit, however they require frequent use as they come in the form of continuous subcutaneous insulin infusions (CSII). This method is therefore mainly utilised by patients with T1DM. Insulin pumps send insulin, through tubing, into an infusion set that delivers insulin to the body. A plastic tube is connected to the insulin reservoir and insulin flows into the subcutaneous tissue via the infusion set. The pumps are very costly and also require commitment to self-management, with frequent blood glucose checks throughout the day. Many adolescents may feel uncomfortable with the concept of being attached to a foreign object. It is also worth noting that insulin pumps do have the potential to over or under medicate patients if they are broken or used incorrectly, and thus potential complications of their use also include ketoacidoses and hypoglycemia. Thus potential complications of use include ketoacidoses and hypoglycemia. 
As there is no definite cure for diabetes, much of the current research on beta cells focuses on developing new and better treatments for diabetic patients. One of the most promising treatments, that is presently being researched and monitored with experimentation, is pancreatic transplantation. Insulin pumps also have the potential to over or undermedicate if they are to malfunction or are used improperly.
1. Pancreatic Islet Transplantation 
This method is specifically aimed to treat patients with type 1 diabetes mellitus. During treatment, the pancreatic islet is isolated within the donated pancreas and the exocrine functioning part of the pancreas is removed. The isolated islet is then transported to the liver of the patient by venous blood.
In a study performed by Shapiro et al. the Edmonton protocol was established and is now used by islet transplant centres around the world, greatly increasing islet transplant effectivity . The protocol involves the islet transplantation in conjunction with immunosuppressive drugs in order to counteract rejection. The procedure of transplantation involves the isolation of islets with cold purified collagenase, which is then digested and purified in a xenoprotein-free medium, and transplanted immediately through a catheter; a small plastic tube using ultrasound for guidance, through the upper abdomen and into the liver (see Figure 11.). This procedure requires only a small dosage of local anaesthetic. Alternatively, some surgeons may create a small incision in patients, with use of general anesthesia. Once implanted, the beta cells in the islet begin to synthesise and release insulin. Ultimately the transplant successfully results in insulin independence with excellent metabolic control.
Currently, many limitations exist with the procedure. These include islet rejection and insufficient supply of islets for transplantation. Although current immunosuppressive regimens exist, they are expensive and may increase the risk of particular malignancies and opportunistic infections. Also as a contradiction, some drugs also impair normal islet function and insulin action, combined with other unwanted side effects, including anemia, weight loss, hypertension, diarrhea and fatigue. Another important concern is renal failure associated with immunosuppressive agents as renal function is a crucial factor in determining the long-term outcomes of patients. With regards to the limitation of islet donors; statistics highlight that at least 1 million americans have T1DM however, only a few thousand pancreas donors are available each year. To resolve this issues researchers continue to look for alternative methods such as growing islets, or at least cells capable of physiologically regulated insulin secretion in vitro.
2. Transplantation using Embryonic Pig Pancreatic Tissue
Instead of using human tissue or organ for transplantation, this method uses the pancreatic tissue of an embryonic pig. Although previous clinical studies failed to provide successful results in treating diabetes, this study has sought to examine the potential and functionality of pig embryonic pancreatic tissues, harvested at different stages of embryonic pig development, in diabetic treatment. In an experimental study, various embryonic pancreatic tissues were implanted in SCID mice and the 42nd day of gestation (E42) showed a substantial growth of pig islets for a prolonged period of time, thereby restoring normoglycemia in the diabetic mice as seen in Figure 12. The study also revealed that reduced tissue rejection was seen at this stage of embryonic tissue implantation, as opposed to E56 or at later stages. Ultimately, the fully immunocompetent diabetic mice, with E42 implanted pancreatic tissue, that were treated with the immunosuppression protocol, attained normal glucose levels. These results thus highlighted the therapeutic potential of E42 embryonic pig pancreatic tissues transplantation in diabetic treatment.
Stem Cell Research
Another promising anti-diabetic therapeutic option is within stem cell research. Mitutsova et al. (2017) investigated the possible use of muscle-derived stem cells (MDSC), from adult mice, to differentiate into functional beta cells in vitro; consequentially compensating for beta-cell deficiency. Within their study they successfully found in vitro cultured MDSC to spontaneously differentiate into insulin-expressing, islet-like cell clusters, under the control of an insulin promoter. These differentiated clusters of beta-like cells co-expressing insulin with transcription factors were shown to secrete significant levels of insulin in response to glucose challenges. Additionally, in vivo undifferentiated MDSC injected into streptozotocin* treated mice, engrafted within 48 hours specifically, to damaged pancreatic islets and were also shown to differentiate and express insulin within 10-12 days after injection. Also to add to the successful results, injection of MDSC into hyperglycemic diabetic mice was shown to reduce blood glucose levels for 2-4 weeks. Ultimately the study successfully showed MDSC as capable of differentiating into mature pancreatic beta like cells, both in vivo and in vitro highlighting a promising avenue in stem-cell based treatment of beta-cell deficiencies.
Generation of Functional Beta-like Cells
In a recent study by Lima et. al. (2016), the generation of mature and functional beta-like cells from the human exocrine pancreas were assessed as a viable avenue for the treatment of type 1 diabetes. The pivotal finding of the study was that the knockdown of transcription factor, ARX, expression, combined with the over-expression of transcription factor, Pax4, considerably enhances the production of functional insulin secreting beta-like cells while suppressing of alpha cells. The beta-like cells produced were shown to efficiently exhibit glucose-responsive insulin secretion. They also showed immediate and prolonged effects in normalising blood glucose levels upon transplantation into diabetic mice.
The study estimated approximately 3 billion of these cells would have an immediate therapeutic effect following implantation in T1DM patients and potentially one pancreas could provide enough tissue for numerous transplants. Thus, the ability to reprogram exocrine pancreas tissue could circumvent the necessity for multiple pancreas donors to treat a single patient. The study, although not achieving exact human islet response levels, did succeed in attaining results of 15-30% insulin levels of that of the normal human islet. This response could potentially have large therapeutic benefits for a patient with T1DM, as reinstating even a fraction of the normal beta-cell mass would be beneficial.
The study thereby produced functional beta-like cells that efficiently processed proinsulin to insulin, contained insulin storage granules, secreted insulin in response to glucose and rescued diabetes in a streptozotocin mouse model. Essentially, this inexpensive and relatively simple protocol, for cell preparation, could provide an avenue to bypass the obstacle of pancreas donors as one donor could provide numerous islet grafts.
Glibenclamide as Potential Prevention of Diabetes
A study by Lamprianou et al. (2016) utilised previous findings on the importance of Cx35 as an enhancement factor for promoting beta cell insulin secretion and resistance to pro-inflammatory cytokines, and extended this research further by analysing the effect of glibenclamide on beta cells. Glibenclamide is shown to promote the assembly and function of Cx36 channels. This study utilised untreated NOD mice to model type 1 diabetes and found the glibenclamide to:
- Protect Cx36 loss in vitro usually induced by Th1 cytokines
- Prevent the development of hyperglycemia through the prevention of beta cell loss (which was shown to rapidly develop in the aging untreated NOD mice)
- Modify the proportion of CD4+ and CD8+ T cells in pancreatic draining lymph nodes
Figure 13. highlights the study's highly successful results, comparing the before and after amounts of islet and beta cell mass. The images of the islets in Figure 13a. highlight the substantial increase in islet number and size after dose 1 and 2 of glibenclamide compared to the control. Figures 13b. and 13c. also highlight the numerical density changes in islet and beta cells respectively, emphasising a spike of approximately 66% islet density and 50% beta cell density after dose 2. The study, therefore ultimately establishes glibenclamide treatment as another plausible avenue for diabetic therapeutics.
RA index to Determine Beta-Cell Functionality
This study aims to identify parameters which influence the function of beta-cells in order to potentially find markers of disease progression or targets for intervention. Although, body mass index (BMI) and increased energy intake are commonly considered risk factors associated with beta-cell dysfunction, evidence of direct numerical correlation remain undefined. Thus, Curran et al. (2016) recognised the necessity for identification markers to allow for beta-cell function assessment.
Beta cells were calculated as a ratio of the incremental insulin to glucose response over 30 minutes of the OGTT. Insulin sensitivity and beta-cell homeostatic assessment of insulin resistance was calculated as well as C-peptide amount. After all else was accounted for, an RA index was formulated as a ratio of resistin to adiponectin at fasting levels. Ultimately the findings showed that the RA index was strongly associated with pancreatic beta-cell function; in vitro studies found direct correlation of RA index to insulin secretion. Previous studies have also found the RA index to coincide with T2DM however this study was the first to mark its connection to beta-cell functionality. The study concluded that there was a necessity for further future investigation into potential mechanisms for modulating the RA index which consequentially may lead to new mechanisms for improving beta-cell function.
Try to attempt these questions from memory!
|Adiponectin||A protein involved in regulating glucose levels as well as fatty acid breakdown.|
|Adipose||Bodily tissue which stores brown or white fat.|
|Alloxan||A toxic glucose analogue which selectively destroys insulin-producing cells in the pancreas when administered in rodents and many other animal species.|
|Anemia||A deficiency of red blood cells.|
|Autocrine||A cell produced substance that has an effect on the cell by which it was secreted.|
|Chrome sublimate||A staining method to identify beta-cell granules (appear as a violet colour).|
|Clathrin-clad granule||Clathrin is a protein that plays a major role in the formation of coated vesicles.|
|Cytoplasm||The material within a living cell excluding the cell nucleus.|
|Edmonton Procedure||Researchers use specialised enzymes to remove islets from the pancreas of a deceased donor and transplant to a patient via catheter through to small incision through to the liver as treatment for type 1 diabetes.|
|Endocrine||Glands that secrete hormones directly into the blood.|
|Endoderm||The innermost layer of cells or tissue of an embryo in early development.|
|Endoplasmic reticulum||Usually has ribosomes attached and is involved in protein and lipid synthesis.|
|Epidemiology||The study of distribution and determinant of diseases.|
|Ergastoplasms||Ribosome-studded endoplasmic reticulum.|
|Etiological||Relating to cause or origin.|
|Exocrine||Glands that secret products through ducts.|
|Fetal position||Back is curved, head is bowed, limbs are bent and drawn up to torso.|
|Fibroblasts||Cells in connective tissue which produces collagen and other fibres.|
|Glucagon||A hormone formed in the pancreas which promotes the breakdown of glycogen to glucose in the liver.|
|Glucokinase||Recognises how high blood glucose levels are in the body.|
|Glycemia||Presence of glucose in the blood.|
|Golgi complex||Also known as golgi apparatus/body is a complex of vesicles and folded membranes within the cytoplasm of most eukaryotic cells, involved in secretion and intracellular transport.|
|Heterologous||Anything other than homologous.|
|Homologous||Having the same relation, relative position, or structure.|
|In vitro||Performed or taking place in a test tube, culture dish, or elsewhere outside the living organism.|
|In vivo||Performed within a living organism.|
|Islets of Langerhans||A cluster of endocrine pancreatic cells, which secrete the hormones insulin and glucagon.|
|Lordotic position||increased inward lumbar spine curvature.|
|Mesenchyme||Mucoid connective tissue consisting of loosely packed, unspecialised cells set in a gelatinous ground substance, from which connective tissue, bone, cartilage, and the circulatory and lymphatic systems develop.|
|Mitochondria||An organelle which carries out the biochemical process of respiration and energy production.|
|Neurosecretory||Storage, synthesis and release of hormones from neurons.|
|NOD mice||Non-obese diabetic mice are used as an animal model for type 1 diabetes.|
|Normoglycemia||a normal concentration of sugar in the blood|
|Notochord||A cartilaginous skeletal rod supporting the body in all embryonic animals.|
|Nucleus||A dense organelle typically a single rounded double membrane structure containing genetic material.|
|Oral Glucose Tolerance Test (OGTT)||Test determining the body's capacity to maintain glucose levels; results noted after overnight fasting and 4 times after administration (usually through sweet liquid) of 75g of glucose (100g for pregnant woman).|
|Pancreas||A large gland within organisms which has an exocrine portion that secretes digestive enzymes and an endocrine portion that secretes the hormones insuln and glucagon.|
|Pancreatectomy||Surgical removal of the pancreas.|
|Pathogenesis||The manner of development of a disease.|
|Phosphorylation||introduction of phosphate group into an organic compound.|
|Plasma glucose||The concentration of glucose in blood measured in milligram per deciliter or milimole per litre. (normal fasting is >100 mg/dL (5.55 mmol/L).|
|Pluipotent||Capable of giving rise to several cell types.|
|Polydipsia||Abnormally increased thirst symptomatic of disease.|
|Polyphagia||Excessive eating or appetite symptomatic of disease.|
|Polyuria||The production of abnormally large volumes of dilute urine ( >2.5L per day).|
|Progenitor||A cell that has a tendency to differentiate into a specific type of cell; like a stem cell.|
|Proliferation||Rapid increase in the number of something.|
|Proteolytic||The break down of proteins into smaller polypeptides or amino acids.|
|Resistin||A cytokine secreted by adipocytes into the circulation causing resistance of peripheral tissues to insulin.|
|SCID mice||Mice severely deficient in functional B and T lymphocytes.|
|Secretory granules||Unique organelle in which neuropeptides and/or hormones are packaged and stored for secretion|
|Somatostatin||Hormone secreted in the pancreas and pituitary gland which inhibits gastric secretion and somatotrophin release.|
|Streptozotocin||A naturally occurring chemical that is particularly toxic to the insulin-producing beta cells of the pancreas in mammals.|
|Sulphonylurea||A class of hypoglycaemic agents taken by people with type 2 diabetes to increase the secretion of insulin by the pancreas.|
|Unmyelinated||Myelin is a fatty white substance that surrounds the axon of some nerve cells, forming an electrically insulating layer. When unmyelinated, the nerve does not have this surrounding layer.|
|Uremic||Uremia is the condition of having urea in the blood; symptomatic of chronic kidney disease.|
|Zymogen||Inactive forms of digestive enzymes.|
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