Isolated Islets and Their Role in Diabetes Research

Introduction

Close to 350 million people worldwide suffers from diabetes in the recent times, out of which about 5% of patients suffer with Type 1 diabetes resulting from absolute loss of insulin production, and ~95% of patients live with Type 2 diabetes and impaired insulin sensitivity as well as with problematic insulin secretion. Islets of Langerhans, present within the pancreas, are miniature organ systems that are responsible mainly for the production of glucagon, insulin, somatostatin and pancreatic polypeptide post appropriate stimulation. Islet research is mainly aimed at curing and/or improving diabetes mellitus which results from a loss of insulin secretion from beta cells or reduced insulin sensitivity of tissues. Since their discovery in 1869, islets have been considered a potential in vitro system for a syndrome that cannot be imitated in a very effective manner using cell lines.¹ Islets are miniature organ systems, preserving their architecture, differentiated status and insulin secretion capacity upon stimulation which is independent of nervous control. Islet isolation has fostered studies on the understanding of the pathophysiology of type I and II diabetes, transplantation and screening of hypoglycemic drugs.^2,3 This review seeks to take an overview of the role of isolated islets as an experimental model in diabetes research.

Role of islets in glucose homeostasis

Langerhans islets are small island-like structures in the exocrine pancreas that represent only 1-2% of the total organ. Five different types of cells contribute to the release of hormones from the islets - α-cells (1520%) secrete glucagon; β-cells (65-80%) produce amylin, C-peptide and insulin; γ-cells (3-5%) secrete pancreatic polypeptide (PP); δ-cells (3-10%) produce somatostatin, and ε-cells (<1%) producing ghrelin.^4-6 These hormones, altogether regulate the homoeostasis of glucose in vertebrates. Glucagon increases blood glucose level while insulin reduces it. Somatostatin prevents both glucagon and insulin release, while PP controls the activity of exocrine and endocrine secretion of the pancreas.^7-9 Although the cellular composition of islets is similar within various species, i.e., humans, rats and mice, their cytoarchitecture differs considerably. While islets of rodents consist predominantly of central β cells and other types of cells in the periphery, human islets are composed of interconnected α- and β-cells.^4,10 The pancreas, through insulin and glucagon, maintains blood glucose levels in a very close range of 4-6 mM. This conservation is achieved through the opposite and balanced actions of glucagon and insulin, known as glucose homeostasis. When blood glucose levels are low during sleep or in between meals, α-cells release glucagon to promote hepatic glycogenolysis. Furthermore, during extended fasting, glucagon stimulates hepatic and renal gluconeogenesis, which raises endogenous blood glucose levels.¹¹ Insulin secretion from β-cells, on the other hand, is triggered by elevated exogenous glucose levels, such as those found after a meal.¹² Insulin initiates the insulin-dependent absorption of glucose into muscle and adipose tissue after binding to its receptor on these tissues, lowering blood glucose levels by eliminating exogenous glucose from the bloodstream.^13-15 Insulin also promotes glycogenesis,^16-18 lipogenesis,^19,20 and the incorporation of amino acids into proteins,²¹ making it an anabolic hormone as opposed to glucagon’s catabolic function.

Islet destruction in diabetes mellitus (DM) and regeneration

Islet destruction: β-cell death in diabetes mellitus

Type 1 and type 2 diabetes are the two most common types of diabetes characterized by progressive β-cell failure. This is usually triggered by an autoimmune attack on the β-cells, which results in progressive β-cell death in type 1 diabetes. Type 2 diabetes (T2DM) has a more varied pathogenesis, with varying degrees of β-cell failure along with varying degrees of insulin resistance. At the time of diagnosis, β-cell mass in type 1 diabetes is reduced by 70-80%. Because of the varying degrees of insulitis and the lack of visible β-cell necrosis, it was hypothesized that β-cell loss happens gradually over time.²² Initial pathological studies in type 2 diabetes people revealed a 25-50 percent loss of β-cells.²³ Apoptosis of beta cells could thus be a common trait of both type 1 and type 2 diabetes. According to recent research, both types of diabetes are characterized by intra-islet production of inflammatory mediators (particularly the cytokine IL-1β), which leads to β-cell death, increasing β-cell loss, and diabetes.^24,25 Invading immune cells create cytokines such as IL-1β, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ in the insulitis lesion of type 1 diabetes. Interleukin-1β  and/or TNF-α plus IFN-γ activate β-cell gene networks under the direction of the transcription factors NF-κB and STAT-1, resulting in apoptosis. NF-κB activation results in the generation of nitric oxide (NO) and chemokines, as well as calcium depletion in the endoplasmic reticulum (ER). Mitogen-activated protein kinases are activated, ER stress is triggered, and mitochondrial death signals are released, all of which contribute to β-cell death. In type 2 diabetes, chronic exposure to high amounts of glucose and free fatty acids (FFAs) induces β-cell dysfunction and may induce death. FFAs may cause β-cell apoptosis through ER stress, which is independent of NF-κB and NO. Thus, cytokines and nutrients cause β-cell death through fundamentally distinct pathways, with cytokines triggering an NF-κB-dependent mechanism that leads to caspase-3 activation and nutrients triggering an NF-κBindependent mechanism.²⁶ De-differentiation of mature insulin-producing β-cells to a “naive” state has recently been identified as a novel mechanism of T2DM-related β-cell failure.²⁷ As a result, the only way to improve insulin-dependent T2DM patients’ treatment is to replace or regenerate lost or defective β-cell mass. This could be accomplished by inducing β-cell proliferation or neogenesis, reversing β-cell dedifferentiation, or preventing -cell apoptosis.

Beta cell proliferation: Genes involved

Islet regeneration refers to the proliferation and replication of existing islet cells to enhance β-cell mass. In the prenatal and neonatal phases, pancreatic β-cells reproduce quickly. After these phases, however, the ability to replicate rapidly deteriorates. Furthermore, the ability to replicate in rodents and humans differs. Cell cycle regulators and circulating soluble substances regulate the proliferation of β-cells. Many mitogenic drugs have been proven to induce β-cell replication in young rats, but not in humans. Insulin-positive β-cells first arise during embryonic development in mice around embryonic day 13.5 and in humans around weeks 8-9. Beta cells are mostly produced during the fetal period by the differentiation of endocrine progenitor cells.²⁸ Beta cells are produced by replicating existing β-cells during the late gestational and neonatal periods. After weaning, the rate of β-cell replication slows, and β-cell renewal capability is reduced until maturity or late adolescence.^29,30 Multiple mitogenic signalling pathways, including IRS-PI3K-Akt, GSK3, mTOR, ChREBP/cMyc, Ras/Raf/Erk, and NFATs, are involved in beta cell replication. These methods also involve upstream activators of mitogenic signalling pathways, including nutrients (glucose, calcium), epidermal and platelet-derived GFs (Glp1, Gip), and hormones (leptin, estrogen, prolactin, and progesterone). Mitogenic signals affect the production of downstream cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), cellcycle inhibitors, and E2F factors, causing quiescent cells to re-enter the cell cycle.^31-36 Systemic regulators that affect β-cell proliferation during puberty, pregnancy, and obesity are circulating soluble substances coming from other organs. The modulation of β-cell proliferation in response to insulin resistance has been linked to a number of circulatory regulators. Intestinal peptides like GlP-1 and GIP-1,^37,38 adipokines generated from adipose tissue like adipsin,^39,40 resistin, and leptin,^41,42 and skeletal-muscle released factors like Il-6 and Il-10 are all examples of circulating regulators.^43,44 Whereas multiple mice studies^45-47 have revealed that β-cells do not proliferate, lineage tracing investigations have confirmed that human β-cells proliferate and give birth to a progenitor/stem cell population. Reg (Regenerating islets derived proteins), HNF-6, SOx9, NeuroD1, Netrin-1 and Neurogenin-3 are among the genes and transcription factors involved in this process.^48-52 Five REG proteins from the Reg gene family have been found in humans so far. In vivo investigations utilizing transgenic and knockout mice have demonstrated that several members of this family are involved in β-cell replication and/or neogenesis.⁵³ In autoimmune type 1 diabetes, they also help to maintain β-cell mass.⁵⁴

Transcription factors in β-cell proliferation

SOx-9, HNF-6, NGN-3, and NeuroD1 are among the transcription factors reported to be involved in the proliferation of β-cells. During pancreatic organogenesis, SOX9 is the first particular marker and maintenance factor of multipotential progenitors. SOx9 increases pluripotent progenitor cell proliferation and inhibits apoptosis in the embryonic pancreas. It regulates pancreatic progenitor cell survival via regulating Notch signalling.⁵⁵ The homeodomain-containing transcrip- tion factor hepatocyte nuclear factor 6 (HNF-6) is an essential regulator of endocrine development. HNF6 is required for maintaining NGN-3 expression and appropriate pancreatic duct shape in the developing pancreas and has both early and late functions.⁵⁶ NeuroD1, a downstream target of NGN-3, continues the endocrine differentiation procedure started by NGN3 and helps the mature islet cells maintain their differentiated phenotype.⁵⁷ NGN-3 determines endocrine cell fate early in pancreatic endocrine development, while NeuroD1 drives endocrine cell differentiation.⁵⁸

Transdifferentiation of pancreas

Islet neogenesis refers to the transdifferentiation of adult pancreatic stem cells, which are thought to be present in the ductal epithelium or acinar tissue, to increase β-cell bulk. The conversion of pancreatic alpha or delta cells into insulin-producing β-cells is known as transdifferentiation. This process is aided by a number of genes and proteins. INGAP, Gastrin, MAFA, PDX1, FOXA2, NKX2.2, NKX6.1, PAX4, and others are among them. Gastrin causes islet-cell neogenesis from pancreatic exocrine duct cells, MAfA reprograms differentiated pancreatic exocrine cells into cells that closely resemble β-cells, and INGAP initiates islet neogenesis. PDX-1 starts endocrine neogenesis, fOxA2 is a major upstream regulator of PDX-1, NKX2.2 regulates pancreatic endocrine differentiation, NKX6.1 keeps and expands the population of β-cell precursors as they progress from precursors to differentiated β-cells, and PAx4 is expressed in endocrine cell progenitors and directs the formation of beta and delta cells.^59-66

Isolation of islets

for diabetic research, islet isolation from the pancreas of mice is a standard procedure. Collagenase perfusion, pancreatic digesting, and islet purification are the three processes of a traditional method. It is a critical step in achieving the necessary number and quality of islet separation. Because of its unique ability to produce digesting enzymes, the pancreas is very timesensitive when it comes to islet separation. As a result, decreasing variance during collagenase perfusion might improve the islet preparation process. Collagenase solution is perfused through the common bile duct. To put a needle into the common bile duct, direct puncture and catheterization through the gall bladder should be employed. Because of the size and length of the duct, performing such a puncture requires some experience, and it can be stressful, especially for inexperienced technicians.⁶⁷

Islet purification

Purification of the islets is another important stage in the intended preparation. To purify islet from collagenasedigested pancreatic tissue, three procedures can be used: ficoll^68-70 and iodixanol gradient separation,⁷¹ filtration,^71-72 and magnetic retraction.^73-74 A successful islet separation procedure includes enzyme-digesting the tissues connecting the islets to the exocrine tissue, isolating islets from non-islet tissue, and culturing isolated islets in an environment that favours cell viability. Because magnetic retraction is only suitable for islet isolation from humans and large animals, most researchers employ one of two methods to obtain islets from the pancreas of mice. The filtration procedure proposed by Salvalaggio et al.,⁶⁷ and the Ficoll method introduced by Bluestone’s group are the two techniques.⁶⁸ Mouse islet preparation was generated using the filtration procedure described by Salvalaggio et al., which included clamping the common bile duct, distending the pancreas with collagenase V, mincing the pancreas with surgical scissors, digesting and handshaking, and finally filtering through a 100 μm nylon cell strainer. This process resulted in an overall purity of 86 percent (before hand-shaking). The Ficoll approach has the drawback of perhaps being poisonous to the isolated islets. Additional cell straining with a 70 μm nylon cell strainer can be used to remove the exocrine cells and keep the bigger islets. Overall purity is estimated to be around 95%. The use of a density gradient to purify islets from acinar tissue is controversial. Because of the nature of pancreatic tissue, purifying islets from acinar tissue, regardless of the approach, is critical. Separation of islets from pancreatic acinar tissue is required because the exocrine pancreas cells emit numerous digesting enzymes. The hypertonic solution sodium diatrizoate, Histopaque (Sigma-Aldrich, St. Louis, MO, USA), is also employed to isolate different cell types. Our lab employed Histopaque 1077 to make the gradients, and a high level of purity was attained with only a few contaminated acinar cells, which were easily removed during the later handpicking of islets.² The final purity of the product is determined by the animal strain and density gradient parameters. Lean rodents, in our experience, produce a purer final preparation than those with greater fat. The outcome of the gradient purification is also strain-dependent, which is consistent with data demonstrating strain-dependent changes in islet isolation.⁷⁵ Prior to culture, a second purification of islets from acinar tissue is frequently required to further boost islet purity. Using a microscope, we detect the islets, then handpick them from one suspension culture dish and place them in a second dish containing a buffered solution or culture medium, such as HBSS or RPMI, respectively. Islets can then be transferred to a dish and cultured overnight in culture media.

Islet yield

The projected islet yield from an isolation method is highly dependent on the rodent’s age and strain. The total islet yield will be influenced by the technician’s expertise as well as the method of isolation employed. With the help of an experienced technician, yields from diverse mouse strains should range from 200 to 400 islets per mouse, with an average of 300 islets per mouse.^3,76 Rats can produce between 600 and 800 islets per animal.⁷⁵ In comparison, the human pancreas is thought to contain around one million islets, and islet equivalents estimate that a normal isolation provides approximately 250,000450,000 islets.⁷⁷

Islet culture conditions

Islet isolation must be followed by suitable culture conditions to ensure that the islets can recover from the shock of collagenase digestion. Islets cultivated with 11 mM glucose have decreased apoptosis rates and improved vitality than those cultured in conditions with both higher and lower glucose concentrations for rodents.⁷⁸ Extended exposure to high glucose produces toxicity, but media with glucose contents significantly below 11 mM can lower islet insulin content and down-regulate critical genes involved in glucose metabolism.^78,79 RPMI 1640 with serum preserves or augments glucose-stimulated insulin secretion (GSIS) in murine islets, according to studies of optimal culture conditions.⁸⁰ In order to avoid competition for nutrients, the recommended islet density for long-term culture is four islets/cm².⁸¹ Islets have time to recover from the arduous process of collagenase digestion after an overnight incubation of 16-20 hours. Prior to completing viability or functional assessment experiments, islet function must be recovered in a sterile incubator at 37⁰C with 5% CO₂ infusion and humidified air.⁸⁰ Rodent islets can maintain glucose sensitivity in culture for at least 1 week with regular medium changes. Changes in mouse islet function can occur in as short as 1 to 4 days in culture, according to studies from human islets⁸⁰ and probably much longer.^82,83

Islet health and morphology

A visual examination of the islets can provide some basic health information. A significant level of acinar or ductal tissue linked to the islets indicates an underdigested islet preparation. The islets in an overdigested islet preparation will have small ducts and few to no acinar cells, as well as rough edges (islets with rough edges may recover following an overnight culture). After an overnight culture in extreme cases of overdigestion, the islets will break into single cells. A normal, healthy islet will have smooth edges and be spherical. Islets appear round and golden brown when observed under a light microscope with a scanning objective lens, with a diameter of 50250 m. These characteristics, particularly the deeper hue of islets in comparison to the comparatively translucent exocrine tissue, makes it possible to identify islets quickly (Figure 1A-C).

Islet functionality: Glucose-stimulated insulin secretion (GSIS)

The ability of pancreatic islets to regulate the release of insulin and other hormones in direct reaction to variations in extracellular glucose concentration is one of their most important characteristics. Because only islet beta cells manufacture and release insulin in the body, this ability characterizes islet function in significant part. Glucosestimulated insulin secretion is thus a widely used indicator of islet function. Islets are cultivated in a ‘low’ glucose concentration, often about 3 mM, to measure the quantity of insulin produced into the media under ‘basal’ or ‘unstimulated’ conditions in order to determine GSIS. Insulin secretion is stimulated when islets are exposed to greater glucose concentrations, such as 11.1 mM (halfmaximal) or >28 mM (maximal). The islet response to glucose stimulation is biphasic, consisting of a fast spike in insulin release (first phase), followed by a slow decrease to a protracted second phase plateau of insulin that lasts for the length of the stimulus.⁸⁴

Islet protecting agents

Isolated islands have been regularly used to investigate the potential of natural products to protect islets. A wide range of natural and synthetic substances have been utilized to screen for islet protecting and insulin secretagogue actions using cultured islets. The aqueous extract of Teucrium polium (Labiatae) was shown to have insulinotropic properties, as it increased insulin production by nearly 135% after a single dose in isolated islets cultivated under high glucose (16 mM).⁸⁵ A significant increase in insulin secretion was seen in a perifusion system in which an exact number of Langerhans islets were exposed to different fractions of Urtica dioica extracts.⁸⁶ Curcumin inhibited phosphorylation of inhibitor of kappa B alpha, which protected islets from cytokine-induced islet death in vitro by scavenging Reactive Oxygen Species (ROS) and normalising cytokine-induced NF-kB translocation (Iκβα). Streptozotocin (STZ) treated mice had higher levels of pro-inflammatory cytokine in their serum and pancreas, but not those pretreated with curcumin before STZ.⁸⁷ Both glucose (8.3 mM) and glibenclamide (0.01 mM)-induced insulin secretion and phosphorylation of ERK1/2 were found to be potentiated by quercetin. Furthermore, quercetin protected β-cell activity and viability against oxidative damage caused by H₂O₂, as well as causing a substantial activation of ERK1/2.⁸⁸ The effects of resveratrol and a possible relationship with SIRT1 in insulinoma INS-1E cells and human islets were examined using resveratrol-cultured insulinoma INS-1E cells and human islets. There was a significant potentiation of glucose-stimulated insulin secretion. This effect was linked to an increase in glycolytic flow, which resulted in more glucose oxidation, ATP production, and mitochondrial oxygen consumption. Upregulation of critical genes for β-cell function, such as GLUT2, glucokinase, PDX-1, HNF-1, and TFAM, was associated with these alterations. Sirt1 overexpression was also found in INS-1E cells after resveratrol administration.⁸⁹ On streptozotocin-treated islets, the aqueous juice of the bitter gourd fruit Momordica charantia L. was evaluated. These changes were linked to upregulation of key genes for β-cell function, such as GLUT2, glucokinase, PDX-1, HNF-1, and TFAM. SIRT1 overexpression was also discovered in INS-1E cells following resveratrol treatment.⁸⁹ The aqueous juice of the bitter gourd fruit Momordica charantia L. was tested on streptozotocintreated islets. It was discovered that it increased insulin production from STZ-islets, decreased lipid peroxidation, and decreased apoptosis, demonstrating its antioxidant and antiapoptotic properties.⁹⁰ On Il1β induced pancreatic islets, the effects of quercetin and its metabolites quercetin 3′-sulfate, quercetin 3-glucuronide, and isorhamnetin 3-glucuronide were investigated. Quercetin suppressed IL-1β-induced IB phosphorylation, NFkB activation, and iNOS promoter activity, as well as reducing IL-1β-induced nitrite generation, iNOS protein, and iNOS mRNA expression. In addition, quercetin dramatically reduced the inhibitory effect of Il-1β on insulin secretion.⁹¹ GSIS was reported to be enhanced by an oleanane-type triterpenoid saponin derived from Momordica tuberosa (Cucurbitaceae), which reduced NO production, lipid peroxidation, and increased islet survival in STZ- and high glucose-treated islets.²

Conclusion

Isolated islets have established themselves as a popular model system in diabetes research. In contrast to complete organism systems, micro organ systems do not require neurological control and modification in terms of stimulation, secretion, transfection, and signalling pathways. This is also a powerful technique for in vitro screening of hypoglycemic drugs and understanding islet destruction and regeneration mechanisms. Although there are limitations to conclusive success, the improvements discussed here indicate that the future is bright.