2017 Group 3 Project

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2017 Projects: Group 1 - Delta | Group 2 - Duct | Group 3 - Beta | Group 4 - Alpha

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Introduction

Background

Beta cells (β cells) are one of the four major cells of the endocrine* portion of the human pancreas. The human pancreas is a composite organ, which extends from the distal foregut endoderm and contains an exocrine* and an endocrine portion. While the exocrine tissue consists of numerous 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 comprises islets of Langerhans, or pancreatic islets (as reviewed by [1]). The islets of Langerhans constitute approximately 1-2% of the pancreas and are dispersed throughout it [2]. They contain multiple endocrine cells; alpha, beta, delta, gamma and epsilon cells, which secrete different hormones; glucagon, insulin, somatostatin, pancreatic polypeptides and ghrelin, respectively [1]. 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 the total islet mass [3].

Currently, it is predicted that adult humans have approximately 2 million islets of Langerhans, contributing to 2% of total pancreatic weight [4]. Within these pancreatic islets, approximately 60-70% of the total islet-cell population can be attributed to beta cells [5]. 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 [6]. The hormone insulin is also synthesised and secreted by beta cells in response to other factors aside from glucose, including nervous stimuli, other hormones and multiple nutrients [4].

Video 1. Introductory Video on the Role of the Pancreas:  

Youtube Link

History

Year Historical Event
1869 The Islets of Langerhans, or Pancreatic Islets, are first discovered by German anatomist Paul Langerhans within rabbits as anatomical formations with no direct connection to the duct but are not given any function. [7] Previous to the discovery, solely recognised as an exocrine organ. [8]
1889 Joseph Von Mering and Oskar Minkowski successfully completed the first pancreatectomy in a dog. Through this, they discovered the removal of the pancreas induced diabetes in the dog thus irrevocable establighing the connection with the pancreas and diabetes. [9]
1893 Edouard Laguesse who discovered the islets in the human pancreas suggested that they were responsible for the internal secretions that regulate glycemia, and named them after their discoverer Langerhans. [10]
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. [9]
1914 Homans noticed that B-cells are involved in experimental diabetes and attributed the sugar regulating function to B-cells. [8]
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. [9]
1943 Dunn et al. selectively destroyed beta cells by administration of alloxan, which led to diabetes. [8]
1957-1974 morphopligical features of the islet cell types were established through multiple studies utilising electron microscopy, including the structure and apppearance of their cytoplasmic secretory granules. [10]
1966 First case of insulin independence by pancreas transplantation by William Kelly and Richard Lillehei into a 28 year old uremic woman at University of Minnesota. [11]
1972 Pictet and Rutter analysed the ultrastructural development of the pancreas by transmission electron microscopy and found both the endocrine and exocrine cells of the pancreas derived from a common progenitor; alpha cells were the first endocrine cells to differentiate. [9]
1977 Cudworth et al. made crucial step in the etiological classification of diabetes; reintroducing terms type 1 and type 2 diabetes. [12]
1990 Sharp et al. led further advances in transplantation achieving clinical insulin independance lasting 1 month through islet transplantation. [13]
2000 Shapiro et al. utilise "Edmonton protocol" where 7 patients with type 1 diabetes receive iselts and all recipients achieve insulin independence. Study utilised Immunosuppression drugs to prevent rejection. [14]
2014 Pagliuca et al. conduct first successful generation of functional human pancreatic beta cells in vitro, providing potentially an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. [15]
Continuing Research continues into stem cell therapeutics for diabetes (refer to "Current Research" below)

Structure

Figure X. Microscopic structure of pancreas

Among most species, pancreatic islets are generally characterised by an oval shape. Nevertheless, the distribution of cells within the endocrine portion of the pancreas is subject to variation. In most mammals, the pancreatic islet contains a core of beta cells, which is encircled by a mantle of non-beta cells. Yet, 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 oval of clover-leaf shaped.

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 such organelles however contain several fenestrae, or openings. While it is not confirmed whether these fenestrae are static or whether they vary in quantity and distribution, it is predicted that they represent areas for communication and 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, called 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 additionally been spotted 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 [5].

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 birth therefore enabling 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, as reviewed by [16].

Structural Comparison between Humans and Mice

Consequential of the clinical importance of pancreas-associated disease, such as diabetes, endocrine tumours and pancreatic cancer, the structure of the pancreas is studied extensively within medical science. Studies of the human pancreas, however, are restricted due to the inherent ethical and medical limitations present in obtaining pancreatic samples at specific and intermittent intervals. As a direct result of scarce human pancreatic samples, animal models are increasingly used in studies. Within current pancreatic research, mice are the most frequently studied animal models. As a sizeable portion of scientific knowledge concerning the cellular architecture of 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.

Fig 1. Comparison of the macroscopic anatomy of the human pancreas (A) against the mouse pancreas (B).
Hand-drawn illustration by user z5158862 comparing the pancreas of the human embryo and the pig embryo.


Macroscopic Comparison

At a macroscopic level, the human pancreas is a well-defined organ, which contains three key structural components: the head, body and tail. 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 touches the hilum of the spleen and 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.

Conversely, the mouse pancreas is less defined than its human counterpart and in a dendritic manner, is uniquely diffused along the mesentery of the proximal small intestine of mice (See Fig. 2.). Analogous to the human pancreas, it can also be distinguished into three key structural components. However, these are called ‘globes’ and are recognized as the duodenal, splenic and gastric lobe. Identified as 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 a component of the splenic lobe, from which it originally develops, and is homologous to the auricle of the human pancreas. Typically, patches of adipose, connective, and lymphatic tissue separate these three main pancreatic lobes in mice.

Microscopic Comparison

Microscopically observed, each lobe of the pancreas contains numerous 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 the 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. The size and distribution of pancreatic islets is congruous between humans and mice. With notably 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 5 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 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 comprise beta cells. In humans, it was originally perceived that alongside a similar mantle-core pattern, beta cells additionally formed ribbon-like clusters and were scattered throughout islets in an unorganised manner. As a consequence of recent studies, however, 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.

Summary Table of Key Comparisons between Humans and Mice
Property Humans Mice
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 [17]). During embryonic day 8.5 and 9, the emerging embryo turns from a lordotic position to a fetal position[18], which causes the endoderm layer to fold on itself and form a primitive gut tube that is divided into the foregut, midgut and the hind-gut. Upon the establishment of the gut tube, the development of the dorsal pancreas is hereafter controlled by the notochord [17], which is the source of permissive signals that enables 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[19]).

Of the three divisions of the gut tube, the foregut is the most essential [20] as the first morphological process in pancreatic formation, and beta cell development, is a condensation of mesenchyme covering the dorsal aspect of the endodermal gut tube in the foregut. Succeeding this condensation, at 26 days post-conception, the endoderm envaginates into the mesenchyme and the dorsal and ventral buds arise from the foregut. The buds are marked by transcription factors, such as pancreatic and duodenal homeobox factor 1 (PDX1) [20], sex-determining region Y (SRY) - box9 (SOX9), and GATA binding protein 4 (GATA4), which are all necessary for pancreatic growth[21]. The epithelium, which constructs the mesenchyme surrounding the pancreas, coelomic epithelium, also begins to compartmentalize 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 [17].

caption

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, acinar enzyme gene expression exponentially increases and the rough endoplasmic reticulum and zymogen granules begin forming, consequently causing the pancreas to become opaque to the naked-eye due to high zymogen granule concentrations.


Although pancreatic alpha cells are 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 [17]. At this stage in development, a down regulation of Pdx1 (pancreatic and duodenal homeobox 1), 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, [22], which is considered to be necessary for proper endocrine differentiation[18]. As the developmental process nears conclusion, endocrine cells aggregate in pancreatic ducts and form islets of Langerhans[17].

On a hormonal level, at around 52 days after the conception of the human pancreas, Neurogenin3 (NGN3), a regulator of pancreatic islet differentiation and regeneration is first detected in the pancreas [23]. Although the expression of the transcription factor Pdx-1 precedes them [22], insulin, glucagon, and somatostatin are the first expressed pancreatic hormones expressed. Pancreatic polypeptide is not detected until about 10 weeks post-conception. At the point where pancreatic islet formation reaches its peak, around 12 weeks into the gestation period, all four hormones are been expressed and the total number of hormone expressing cells, present within the islets, has increased [24].

Function

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 the hormone 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, muscles and adipose tissue. Insulin release into the bloodstream predominantly occurs as a consequence of increased blood glucose levels. Glucose phosphorylation, by glucokinase, is currently understood to act as the glucose sensor, as it adjusts the rate of 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:  

Youtube Link

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 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 [25].

Insulin Biosynthesis

In the 1950s, insulin (as show in Fig. x) 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 retiticulum (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 [REF] and cleaved to form equimolar amounts of insulin and connecting polypeptide (C-peptide) [26]. 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 becomes mature C-peptide and insulin with the action of pro-protein convertase-2 (PC2) and carboxypeptide-E (CPE). The alternative pathway which occurs less commonly is where the 2 amino acids are removed first (see Figure X) [27].

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 [28]). 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.

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.

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, as indicated by nuclear magnetic resonance (NMR) imaging.

Image Proinsulin and Image insulin

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 partly 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 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 (see Fig y.) [26].

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, why the latter phase involved the continuous release of insulin, which slowly increased in rate until the termination of the glucose stimulus [29].

This was the first indication that insulin release from beta cells follows a biphasic pattern [30]. 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 the 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 [31]. 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 are 0.14%/min and 0.05%/min, respectively [30].

Signalling

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 [32].

Caption

In pancreatic beta cells, an increase in blood glucose levels blocks 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[33]. 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[34]). 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 [30].

Conversely, as the electrical activity induced in beta cells is mainly due to calcium ion influx [35], decreased extracellular levels of calcium ions prevent the firing of action potentials and thus, by extension, inhibit insulin secretion [32].

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 replication the effect of glucose in order to block ATP-sensitive potassium channels [32].

While increased glucose levels generate a high cytoplasmic concentration of ATP and depolarize beta cells by blocking ATP-sensitive potassium channels[36], 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 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[32].

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[32], which produces approximately 95% of the cell’s energy and is therefore colloquially termed “powerhouse of the cell” [37]. Within the mitochondria, the electron transport chain (ETC) is the central machinery responsible for ATP production (reviewed in [38]) and is heavily reliant upon a proton gradient across the mitochondrial membrane[32].

The ETC is composed of four enzyme complexes that 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[39] (as shown in Figure. X and further explain in Video 3.).

caption

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 [32] and therefore inhibits the necessary formation of a proton gradient. The overexpression of UCP2 can therefore bypass the production of ATP, which is reinforced by studies that report a 50% reduction in the ATP content in the cells of UCP-2-induced pancreatic islets [40]), 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 [41]). It has also been discovered that the expression of Sirt1in beta cells downregulates UCP2 expression, further enhancing insulin secretion [32].

Video 3. Explanation of the Mitochondrial Electron Transport Chain:  

Youtube Link

Role in Pathology

To reiterate the sections above, insulin is vital in the maintenance of glucose levels. Through signalling of liver, muscle and fat cells, insulin allows for the uptake of glucose, allowing for its use as an energy source. When the body has a sufficient amount of energy, insulin signals the liver to take up 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. Refer to the video below for a visual conceptual explanation of the relationship between insulin, glucose and diabetes.

Video 4. Video to understand the connection between insulin and diabetes:  

Youtube Link

Disease

Type 1 Diabetes Mellitus (T1DM) Type 2 Diabetes Mellitus (T2DM)
Epidimiology

Type 1 diabetes is one of the most common chronic childhood diseases

  • It appears two peaks occur in the presentation of type 1 diabetes; between the age of 5-7 and the other occurring near puberty. [42]
  • accounts for approximately 5-10% of the diabetic population worldwide. [43]

Type 1 diabetes varies dramatically on a global scale with more than 350-fold variation in incidence among reporting countries; overall:

  • least common in China, India and Venezuela
  • most common in Finland as well as Sweden, Norway, Portugal, Great Britain, Canada and New Zealand. [44]
  • The number of type 2 diabetes is increasing with 80% living in low and middle income countries.
  • It is estimated that 439 million people would have type 2 diabetes by the year 2030 [45].
  • The incidence varies substantially from one geographic region to the next due to environmental and lifestyle risk factors [46].
Cause

Type 1 diabetes results from an insulin deficiency that is caused by the loss of pancreatic beta cells

  • consequence of autoimmune destruction of beta cells that generally occurs over an extended period of time
  • Genetic susceptibility is believed to be a prerequisite for type 1 diabetes. [43] However in a study by Barnett, Leslie & Pyke (1981)
  • However type 1 diabetes does not fit any simple inheritance pattern and is considered a complex multifactorial disease. [47]

Early familial and twin studies supported the importance of both genetic and environmental risk factors. [48]

  • the analysis of 200 pairs of identical twins within different age groups revealed approximately half the pairs were discordant'; suggesting type 1 diabetes was not entirely genetically based.
  • An environmental or alternative non-genetic cause was suggested as twins that were living separately between the ages of 20-39 were shown to have a greater number of discordance.[48]
  • Pancreatic beta-cell dysfunction and failure results in the prevalence of type 2 diabetes.

In the normal healthy body, beta-cells are able to function at a greater capacity when glucose increases, thus producing more insulin to maintain glucose homeostasis; beta cells are able to adapt resulting in a compensatory response in insulin production. Beta-cell dysfunction is seen inType 2 diabetes as the compsenatory response results in gradual failure of beta cells, primarily due to insulin resistance within the individual [49]

Primarily lifestyle factors cause this insulin resistance to occur;

  • physical inactivity,
  • sedentary lifestyle,
  • cigarette smoking and generous consumption of alcohol;
  • obesity contributing to 55% of cases. [50].

Recent increase in occurence of type 2 diabetes may also be attributed to environmental factors as reveiwed by Olokoba et al. (2012) [51].

Symptoms
  • Polyuria,
  • increased thirst,
  • unexplained weight loss,
  • blurred vision,
  • fasting plasma glucose of >126mg/dL (7.0mmol/L) ,
  • two hour postload plasma glucose of >200mg/dL (11.1 mmol/L) during standard oral glucose tolerance test (OGTT) [52]
  • Polyuria,
  • polydipsia,
  • polyphagia,
  • raised fasting plasma glucose >126mg/dL (7.0mmol/L),
  • two hour postload plasma glucose of >200mg/dL (11.1 mmol/L) during standard oral glucose tolerance test (OGTT) ( as reviewed by Olokoba et al. 2012) [51]
Pathogeneis

The pathogenesis of autoimmune type 1 diabetes has been extensively studied through the use of animal models; nonobese diabetic mouse and diabetes-prone BioBreeding rat. Exact mechanisms of initiation and progression remain unclear, however through the animal studies it is generally believed that beta-cell autoantigens macrophages, dendritic cells, B lymphocytes, and T lymphocytes are involved in the beta-cell specific autoimmune process. As seen in Figure 3, the beta-cell autoantigens are processed by the macrophages, dendritic cells or B cells in the pancreatic islets, and then presented to the autoreactive CD4+ T cells in the peripheral lymphoid system. These cells are consequently activated leading to the secretion of cytokines which can activate beta-cell specific cytotoxic CD8+ T cells. Activated T cells recruit at the iselts, producing cytokines which further activate macrophages and other T cells; all contributing to the destruction of pancreatic beta-cells. [53]

File:Beta Cell Destruction.JPG
Figure 3: Beta-cell destruction by autoimmune processes

The evolution of beta-cell dysfunction is marked by 5 stages as identified by Weir & Bonner-Weir (2004) [54]:

  • Stage 1: Referred to as compensation; when the insulin secretion must compensate for degrees of insulin resistance for the maintenance of glucose homeostasis.
  • Stage 2: Where fasting levels range between 5-7.3mmol/L, representing 'beta-cell adaptation'. People progressing toward Type 2 diabetes may remain here for many years, however when beta-cell mass becomes insufficient glucose levels may rise to stage 4 rapidly.
  • Stage 3: Early decompensation: glucose levels rise beyond 7.3mmol/L
  • Stage 4: Stable decompensation: glucose levels range between 16-20mmol/L
  • Stage 5: Severe decompensation: extreme beta cell failure; ketosis with blood glucose levels above 22mmol/L
Figure 4:Progression of beta-cell dysfunction

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 any other pancreatic trauma leading to loss of pancreatic tissue; the most common cause being chronic pancreatits (CP). CP accounts for 75-80% of T3cDM cases while pancreatic carncinoma accounts for 8%. [55] T3cDM was previously believed to have a lower prevalence of approximately 0.5-1.2 percent among diabetes cases in North America, however recent studies suggest higher occurences with a recent 2013 study finding a 5-10% prevalece among all diabetic subjects in Western populations. [56] A review by Anderson et al. (2013) suggest previous underestimations may be attributed to the fact that it has become easier to detect exocrine pancreatic pathology with the advancements in imaging technology and noninvasive screening methods to quantify exocrine dysfunction. [57]

Also unlike T1DM and T2DM the endocrinopathy is far more complex in T3cDM due to additional comorbidties. [58] Due to the cross over in risk factors for T2DM and T3cDM such as obesity and the similar symptom associated with all three forms of diabetes; difficulties with glucose homeostasis, a method of discrimination between the diseases is required.

Proposed criteria:

  • presence of exocrine pancreatic insufficency (monoclonal fecal elasttase-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 even with these criteria in place, overlapping still occurs as people suffering from T1DM and T2DM for an extended period of time are also associated with 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 seems to be the pancreatic polypeptide (PP) response to nutrients. [59]

A study found that isolated hepatic insulin resistance coexisted with clinical studies of T3cDM [60]. This has been documentated to be due to CP, pancreatic cancer and cystic fibrosis resulting in unsuppressed hepatic glucose production. PP deficiency was also found to coincide and thus was identified as a likely mediator of the hepatic defect. Animal studies confirmed that PP deficiency results in diminished hepatic insulin receptor availability and thus PP administration reversed hepatic insulin resistance in patience with CP, resulting in improved glucose tolerance. [61]

Figure 5: The effect of adrenomedulin on insulin secretion

How cancer induces T3cDM

The most likely explanation for the consequential occurence of diabetes in pancreactic ductal adenocarcinoma (PDAC) 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 overexpressed in PDAC. Adrenomedullin receptors are found on beta cells, which studies have found with the external administration of adrenomedulin in animal studies cause the inhibition of insulin secretion. [62] 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 5) [63]. These finding support that adrenomedulin is mediator of beta cell dysfunction in PDAC.


How chronic pancreatitis induces T3cDM

Development of diabetes in chronic pancreatitis mainly occurs due to the destruction of islet cells by pancreatic inflammation. Additionally maldigestion caused by pancreatitis leads to diminished insulin release. [64] 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. [65] Thus maldigestion leads to an impaired incretin secretion and therefore diminished insulin release of the remaining beta-cells. [64] In contrast to auto-immune 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.

Treatments

Insulin Therapy

Insulin injections are used in place of the insulin that would normally be produced by the body. Individualised treatment plans are suggested however generally T1DM patients would take 3-4 injections per day to provide optimal glucose control, while T2DM generally would take 2 injections per day if not in conjunction with medication. [66][67] Review Article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901025/#!po=72.5000 Insulin shots are most effective when administered when food enters the blood such as 30 minutes before eating. Also particular sites of injections provide better results, such as the addomen, while insulin taken at the arms and thighs take a longer period of time to arrive into the blood stream. [68]

However insulin vial and syringes provide an inconvenience to patients, providing adverse psychological and social impact in using a syringe as well as incorrect insulin use and dosage could also result in risks. Insulin pens were thus developed to counter these issues, providing rapid, long acting insulin. [69] On the other hand, insulin pens become more costly per unit of insulin compared to the insulin supplied in vials. [68]

Insulin pumps are also another alternative to provide insulin however it is in the form of continuous treatment; also known as continuous subcutaneous insulin infusion (CSII). This method is 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 reservour and insulin flows into the subcutaneous tissue through the infusion set. However pumps are not only costly but require commitment to self-management with frequent blood glucose checks throughout the day. Many adolescents also may feel uncomfortable with the notion of being attached to a foreign object.


Pancreatic Transplantation

As there is no definite cure for diabetes, many of the current research on Beta Cells focus on developing new and better treatments for patients with diabetes. One of the most promising treatments that is still under research and experimentation is pancreatic transplantation. [70] Insulin pumps also have the potential to over or undermedicate if they are to malfunction or are used improperly. Thus potential complications of use include ketoacidoses and hypoglycemia. [71]

The development of more physiological routes of insulin administration, use of artificial pancreas or pancreas/beta cell transplantation thus become important for ongoing research.

1. Pancreatic Islet Transplantation [72]

This method is specifically aimed to treat patients with type-1 diabetes mellitus. The pancreatic islet is responsible for the endocrine function of the pancreas. This is isolated within the donated pancreas, and the exocrine functioning part of the pancreas is removed. The isolated pancreatic islet is then transported to the liver of the patient by the blood of the vein.

Figure 6: Diagrammatic representation of islet transplantation

In a study performed by Shapiro et al. the Edmonton protocol was established which was taken on by islet trancplant centers around the world and has greatly increased islet transplant. [72] 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 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 6). This procedure only requires a local anesthetic, Alternatively some surgeons will create a small incision using general anesthetics. Once implanted, the beta cells in the islet begin to make and release insulin. Ultimately the transplant succefully results in insulin independence with excellent metabolic control. [14]

However currently many limitations remain with this procedure, two of which include islet rejection and supply of islets for transplantation. Although current immunosuppressive regimens exist, they are expensive and may increase the risk or 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 such as anemia, weight loss, hypertension, diarrhea and fatigue. [14] More importantly are the concerns of renal failure associated with umminnosuppressive agents, as renal function is a crucial factor in determining long-term outcomes for the patients. [73] As for the limitation of islet donors; statistics highlight at least 1 million american have T1DM however only a few thousand pancreas donors are available each year. To resolve this issues researchers continue to look for alternative means such as growing islets, or at least cells capable of physiologically regulated insulin sectrion - in vitro.

2. Transplantation using Embryonic Pig Pancreatic Tissue [74]

Instead of using human tissue or organ for transplantation, this method uses pancreatic tissue of an embryonic pig. Although previous clinical studies failed to provide successful results in treating diabetes, this study sought to examine the potential, functionality and immunogenicit of pig embryonic pancreatic tissue harvested different stages of the embryonic pig development.

Through the use of implantation of the varying embryonic pancreatic tissues in SCID mice, the 42nd day of gestation (E42) enabled a substantial growth of pig islets for a prolonged period of time and restored noromoglycemia in the diabetic mice as seen in Figure 7. Also the study reveal reduced rejection was seen at this stage of embryonic tissue implantation, as opposed to E56 or later stages. Ultimately the fully immunocompetent diabetic mice with E42 implanted pancreatic tissue that were simultaneously treated with immunosuppression protocol attained normal glucose levels and elimated the need for insulin.

Thus these results highlight the theraputic potential of E42 embryonic pig pancreatic tissue transplantation in diabetes.

Figure 7: Comparing implantation of varying stages of pig embryonic pancreatic tissue on diabetic mice


Stem Cell Research

Another recent endeavour into a promising anti-diabetic therapeutic is within stem cell research. has also been conducted in the search for anti-diabetic therapeutics. Mitutsova et al. (2017) investigated the possible use of muscle-derived stem cells (MDSC) from adult mice to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate 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. Differentiated clusters of beta-like cells co-expressing insuling 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 differenctiating into mature pancreatic beta islet-like cells, both in vivo and in vitro highlighting a promising avenue in stem-cell based treated of beta-cell deficiencies. [75]

Current Research

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 was assessed for being a viable avenue for treatment of type 1 diabetes. The pivotal finding of the study was that knockdown of transcription factor ARX expression combined with over expression of transcription factor Pax4 considerably enhances the production of functional insulin secreting beta-like cells with the accompanying suppression of alpha cells.The beta-like cells produced were shown to efficiently exhibit glucose responsive insulin secretion and an immediate as well as prolonged effect 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.

The ability to reprogram exocrine pancreas tissue could circumvent the necessity for multiple pancreas donors to treat a single patient, allowing one pancreas to treat several patients instead. The study although could not achieve 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 therapeutic benefits for a patient with T1DM as reinstating even a fraction of the normal beta-cell mass would have a therapeutic effect.

Ultimately this study 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. Essential this inexpensive 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. [76]

Glibenclamide as Potential Prevention of Diabetes

A study by Lamprianou et al. (2016) utilised previous study's findings in the importance of Cx35 as an enhancement factor of insulin secretion, promoting resistance of beta cells against pro-inflammatory cytokines, and furthered this by studying whether glibenclamide could protect beta cells since it is shown to promote the assembly and function of Cx36 channels. The study utilised untreated NOD mice to model type 1 diabetes, and found the glibenclamide to:

Figure X: The Therapeutic Effect of Glibenclamide on NOD Mice
  • protect Cx36 loss in vitro usually induced by Th1 cytokines
  • prevent the development of hyperglycemia through the prevention of loss of beta cells, shown to rapidly develop in the aging untreated NOD mice
  • modified the proportion of CD4+ and CD8+ T cells in pancreatic draining lymph nodes

Figure X highlights the highly successful results, comparing the before and after of islet and beta cell mass. The images of the islets in Figure Xa highlight the substantial increase of islet number and size after dose 1 and 2 of glibenclamide compared to the control. Figure Xb and c 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.

Ultimately the study establishes a plausible avenue for therapeutics as seen through the testing of the therapeutic value of glibenclamide effects. [77]

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 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. Ultimately after all else was accounted for a RA index was formulated as a ratio of restin to adiponectin at fasting levels. Ultimately the findings were that RA index was very strongly associated with pancreatic beta-cell function; in vitro studies correlated RA index to insulin secretion. Previous studies have found the RA index to correlate to T2DM however this study was first to make the connection to beta-cell function.

The study concluded with the venture to examine potential mechanisms for modulating the RA index which consequentially may lead to new avenues/interventions for improving beta-cell function. [49]

Review Quiz

Try to attempt these questions from memory!

1

In which species does the pancreatic islet contain a core of beta cells surrounded by a mantle of non-beta cells?

Most humans and primates
Most mammals
Neither species

2

What is the name of the focal point where adjacent beta cells connect to each other along their plasma membrane?

Islets of Langerhans
Desmosomes
Rough endoplasmic reticulum

3

Which of the following statements is correct?

The proteolytic processing of proinsulin may be prevented by energy poisons, which block their transfer into secretory vesicles
Proinsulin and insulin are highly dissimilar and demonstrate large differences in solubility and isoelectric point
Type 2 diabetes is one of the most common chronic childhood diseases

4

How does an individual become at risk of Type 3c diabetes?

Through genetic predisposition leading to autoimmune attack of beta cells
As a secondary effect from some alternative disease effecting pancreatic tissue and sub-sequentially beta cells
Through a sedentary life style

5

How can beta-like cells become useful in the future of therapetics?

Cost effective treatment option
Less donors required
Simple protocol
All of the above

6

Polydipsia is a defining symptom of which beta-cell related disease?

Type 1 Diabetes
Type 2 Diabetes
All the above

7

The correct definition of polyphagia is:

Excessive eating or appetite symptomatic of disease
The production of abnormally large volumes of dilute urine ( >2.5L per day)
Abnormally increased thirst, symptomatic of disease


Glossary

Term Definition
Adipose Bodily tissue which stores brown or white fat
Edmonton Procedure Researchers use specialised enzymes to remove islets from the pancreas of a deceased donor and transplant to patient via catheter through to small incision through to liver as treatment for type 1 diabetes.
Epidemiology The study of distribution and determinant of diseases
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
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
Pathogenesis The manner of development of a disease
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).
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)
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).
Streptozotocin A naturally occurring chemical that is particularly toxic to the insulin-producing beta cells of the pancreas in mammals

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