Isolated Islets in Diabetes Research

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Isolated islets in diabetes research

R. Bhonde, R.C. Shukla, M. Kanitkar, R. Shukla, M. Banerjee & S. Datar

Tissue Engineering & Banking Laboratory, National Centre for Cell Science, Pune, India

Received November 8, 2006

This review highlights some recent developments and diversified applications of islets in diabetes

research as they are rapidly emerging as a model system in biomedical and biotechnological research.

Isolated islets have formed an effective in vitro model in antidiabetic drug development programme,

screening of potential hypoglycaemic agents and for investigating their mechanisms of action. Yet

another application of isolated islets could be to understand the mechanisms of cell death in vitro

and to identify the sites of intervention for possible cytoprotection. Advances in immunoisolation

and immunomodulation protocols have made xeno-transplantation feasible without

immunosuppression thus increasing the availability of islets. Research in the areas of pancreatic

and non pancreatic stem cells has given new hope to diabetic subjects to renew their islet cell massfor the possible cure of diabetes. Investigations of the factors leading to differentiation of pancreatic

stem/progenitor cells would be of interest as they are likely to induce pancreatic regeneration in

diabetics. Similarly search for the beta cell protective agents has a great future in preservation of

residual beta cell mass left after diabetogenic insults. We have detailed various applications of

islets in diabetes research in context of their current status, progress and future challenges and

long term prospects for a cure.

Key words Cytoprotection - hypoglycaemics - islets - regeneration - transplantation

Islets of Langerhans are organelles present within

the pancreas and are mainly responsible for the

production of insulin, glucagon, somatostatin andpancreatic polypeptide upon stimulation. The primary

focus of islet research is, however, the cure and/or

better management of diabetes mellitus which results

from a loss of insulin secretion from beta cells present

within the islets of Langerhans. This review seeks to

take a bird’s eye view of the contribution of islets as a

model system in diabetes research beyond

transplantation.

Since their discovery in 18691, islets have been

viewed as a possible in vitro system for a syndrome

that cannot be mimicked very effectively using celllines. They are miniature organ systems, retaining their

architecture, differentiated state and ability of insulin

secretion upon stimulation, independent of nervous

control. Isolation of islets2,3 has promoted studies related

to understanding the pathophysiology of type I and II

diabetes, transplantation, screening of hypoglycaemic

drugs and probing into diabetes causing mechanisms,

to device effective means of prevention.

425

Indian J Med Res 125, March 2007, pp 425-440



Islets in pharmacological research

Insulin secretion enhancers: Isolated islets, in vitro,

respond to glucose stimulation and hence haveimmensely contributed to the study of various

pharmacological aspects and for screening of

promising antidiabetic agents. A number of

hypoglycaemic drugs act as insulin secretagogues,

in corroboration with this, we have reported that islets

can serve as an in vitro model for antidiabetic drug

screening4. Our model offers several advantages as

it is simple and economical in terms of reduction in

the number of animals used as well as the amount of

drug required for testing. A number of plants, in

traditional medicine, are claimed to have anti-

diabetic properties and isolated islets have been used

extensively for checking their properties and

plausible modes of action. For example, extract of

Tinospora crispa5, extract of Gymnema sylvestre6,

bittergourd fruit juice7, leaf extract of Urtica diocia8,

aqueous extract of Scoparia dulcis9 and aqueous

extract of Teucrium polium10 have been shown to

exhibit insulin secretagogue activity. Apart fromplant/natural extracts, several synthetic drugs have

been tested for their insulin secreatagogue property

and for determination of their mechanism of action,

using isolated pancreatic islets in vitro.

It is understood that glucose stimulates insulin

secretion in the pancreatic cell by means of a

synergistic interaction between at least two signaling

pathways. In the K (ATP) channel-dependent

pathway, glucose stimulation increases the entry of

extrinsic Ca2+ through voltage-gated channels by

closure of the K (ATP) channels and depolarization

of the beta cell membrane. The resulting increase in

intracellular Ca2+ stimulates insulin exocytosis.

While in the GTP-dependant pathway, intracellular

Fig. 1. Signaling cascade of glucose stimulated insulin secretion. The figure summarizes the signaling pathway triggered by glucose

in beta cells for insulin exocytosis. Agents in blue represent secretagogues while agents in red represent insulin secretion inhibitors.

Gq protein: member of G family of proteins, IP3: Inositol triphosphate.

426 INDIAN J MED RES, MARCH 2007



Ca2+ is elevated by GTP-dependent proteins and

augments the Ca2+-stimulated release (Fig. 1).

Secretagogues and insulin secretion inhibitors act at

intermediate steps of these signaling pathways andinfluence the process of insulin exocytosis. Several

researchers have investigated this intricate mode of

known secretagogue action using isolated islets as

an in vitro model. To quote a few; imidazoline

antagonists of alpha 2-adrenoreceptors increase

insulin release in vitro by inhibiting ATP-sensitive

K+ channels in pancreatic cells11. Giannaccini et

al12 have evaluated the properties of sulphonylurea

receptors (SUR) of human islets of Langerhans. They

studied the binding affinity of various oral

hypoglycaemic agents to the receptor and also tested

insulinotropic action of the drugs on intact human

islets. This binding potency order was parallel with

the insulinotropic potency of the evaluated

compounds12 . Masuda et al13 have shown an

insulinotropic effect of Triglitazone (CS-045) and

have shown its mode of action to be distinct from

glibenclamide (a sulphonylurea drug). A-4166, a

derivative of D-phenylalanine, evokes a rapid and

short-lived hypoglycaemic action in vivo. It has been

shown to act via the tolbutamide binding sites 14.Louchami et al15 showed S21403, a meglitinide

analogue to be a novel insulinotropic tool in the

treatment of type 2 diabetes, as it affected cationic

fluxes and the drugs secretary responses displayed

favourable time course of prompt, and not unduly

prolonged, activation of cells. Mears et al16

demonstrated that tetracaine (an anaesthetic)

stimulates insulin secretion by release of intracellular

calcium and for the first time elucidated the role of

intracellular calcium stores in stimulus-secretion

coupling in the pancreatic cells. JTT-608, is a

nonsulphonylurea oral hypoglycaemic agent which

stimulates insulin release at elevated but not low

glucose concentrations by evoking PKA-mediated

Ca2+ influx17.

Insulin secretion inhibitors: Besides insulinotropic

studies, several investigators have used isolated islets

to study drugs that inhibit insulin secretion and their

probable mode of action. Several drugs have

inhibitory effect on insulin secretion by cells and

this is an important aspect, which needs to be

considered while using the said drug. Cyclosporine,

a widely used immunosuppressant, induces inhibitionof insulin release in isolated rat islets18,19. Paty et al20

demonstrated the inhibitory effects of several

immunosuppressive drugs on insulin secretion. Thus

low dose immunosuppressive drug protocols should

be used in clinical islet transplantation and patients

using this drug have to be carefully monitored for

signs of deficient insulin secretion. Tacrolimus is

known to cause post-transplant diabetes mellitus.

Using isolated rat pancreatic islets, Uchizono et al21

studied its mechanism of action and found that it

interferes with the process of insulin exocytosis and

that protein kinase C (PKC)-mediated (Ca2+

dependent and independent) and Ca2+-independent

GTP signaling pathways may be involved. L-

asparaginase inhibits glucose-induced insulin

secretion in a dose-dependent manner by decreasing

total cAMP in isolated rat islets22. Metz et al23 have

used selective inhibitors of GTP synthesis and proved

that they impede exocytotic insulin release from

intact rat islets. The study provides first direct

evidence that GTP is required for insulin release23.Hydrochlorothiazide is also known to inhibit insulin

release. This inhibition is not mediated by reduced

chloride fluxes but rather by inhibition of calcium

uptake24.

Many antidiabetic drugs act on peripheral tissues

and have no direct effect on pancreatic islets. One

such drug is metformin, which affects glucose and

free fatty acid metabolism in peripheral insulin target

tissues. However, Patane et al25 exposed rat

pancreatic islets to high glucose and free fatty acid

(FFA) levels in vitro mimicking the in vivo

environment in presence and absence of metformin.

They found that metformin can restore a normal

secretary pattern in islets whose secretary function

has been impaired by chronic exposure to elevated

FFA or glucose levels. Recently, Marchetti et al26

cultured islets isolated from type 2 diabetes subjects

in presence of metformin and found that several

functional and survival defects of T2D islets were

BHONDE et al: ISLETS IN DIABETES RESEARCH 427



ameliorated by metformin. Thus in diabetic patients,

metformin (in addition to its peripheral effects) may

have a direct beneficial effect on the cell secretory

function. On the contrary, Leclerc et al27 have shownthe ability of metformin to activate AMP-activated

protein kinase in human islets and inhibit insulin

secretion. This inhibitory effect needs to be

considered with respect to the use of this drug for

the treatment of type II diabetes.

We examined the effect of commonly used

antibiotics such as gentamycin, penicillin,

streptomycin, tetracycline, neomycin, erythromycin

and chloramphenicol on isolated islets viability,

functionality and induction of oxidative stress if any.

Our results revealed the innocuous nature of the

antibiotics used at pharmacological concentrations,

suggesting their safety whenever prescribed for

combating infections and also during islet isolation

procedures28.

Cytoprotection of islets

Another primary area of research greatly

facilitated by usage of islets as a model system is inunraveling the mysteries of beta cell death and ways

of preventing the same.

cell death: The precise mechanisms of cell death

in vivo, leading to diabetes, remain unclear. However,

extensive studies, using islets as a model system show

that there are many molecules including Fas Ligand

(FasL) and cytokines such as interleukin-1 (IL-1),

tumour necrosis factor alpha (TNF) and interferon

gamma (IFN ) that cause release of other cytokine

mediators that have potential to damage cells in

vitro and in vivo29. cell death appears to be

ultimately caused by receptor mediated mechanisms

and/or by secretion of cytotoxic molecules like

granzymes and perforin. In addition, toxic molecules

such as reactive oxygen species (ROS: superoxide

radicals, hydroxyl radicals, and nitric oxide) play a

significant role in islet cell death by inducing DNA

damage. DNA damage, in cells, leads to poly (ADP

ribose) polymerase (PARP) activation which

increases NAD consumption, depletion of which

compromises ATP production in cells30. It is apparent

that a number of different mechanisms of cell death

are operative in destruction of islets (Fig. 2).

Inhibi tion of ox idants : Normally, cells counter

oxidative stress by expression of ROS scavenging

enzymes like catalase (CAT), glutathione peroxidase

(Gpx) and superoxide dismutase (SOD). cells,

however, have extraordinarily low levels of ROS

scavenging enzymes31 . The correction of this

deficiency, in vitro, by overexpression of cellular

enzymes like SOD may lead to protection of cells

against oxidative stress induced cell damage/

death32. Studies, wherein mitochondrial form of Mn-

SOD was overexpressed in cells are shown to have

protected isolated islets against oxidative damage in

vitro33. It has also been shown that adenoviral

overexpression of glutamyl cyesteine ligase catalytic

subunit, a primary regulator of de novo synthesis of

glutathione (GSH) in mammalian cells and central

to the antioxidant capacity of the cell, protects

pancreatic islets against oxidative stress, in vitro34.

In vitro stress in islets is often produced by using

cell specific toxins like streptozotocin (STZ) andalloxan. STZ induces islet necrosis by employing

effector molecules like nitric oxide (NO) and ROS.

Pro-inflammatory cytokines also employ NO as an

effector molecule for necrosis and / or apoptosis

induction 35 . Hence a plausible means for

cytoprotection of islets could be scavenging of NO

or inhibition of iNOS (inducibe nitric oxide synthase)

which synthesizes NO. iNOS inhibitors can be used

for cytoprotection of islets in vitro as inhibition of

iNOS would inhibit formation of NO, preventing islet

cell death indirectly. It has been reported that a

combination of an iNOS inhibitor and a free radical

scavenger, guanidinoethyldisulphide restored IL-1

induced suppression of islet insulin secretion in

vitro36. An imidazole compound called Efaroxan has

also been shown to impart complete protection

against IL-1 induced toxicity37. It has recently been

shown that silymarin, a polyphenolic flavonoid that

has a strong antioxidant activity, prevented IL-

1+IFN- -induced NO production and -cell




dysfunction in human islets. These cytoprotective

effects of silymarin appeared to be mediated through

the suppression of c-Jun NH2-terminal kinase and

Janus kinase/signal transducer and activator of transcription pathways38. In vitro treatment of islets

with exogenous antioxidants is another viable option.

There are many known antioxidants like vitamin C,

vitamin E39, Scoparia dulcis a traditional antidiabetic

plant40 that are known to protect islets against

oxidative stress induced dysfunction and death.

Studies with islets have shown that the experimental

drug, bis-o- hydroxycinnamoyl methane, an analogue

of naturally occurring bis- demethoxycurcumin,

enhances the antioxidant defense against

ROS, thus protecting cells from death41. It has been

demonstrated that polyenoylphosphatidylcholine

(PPC), a phosphatidylcholine rich phospholipid

extracted from soybean, protects cells against STZ

induced toxicity and also plays an important part in

maintaining their insulin synthesis and secretion for

normal glucose homeostasis42.

PARP and NF B inhibition : Poly (ADP ribose)

polymerase (PARP) is a major effector molecule in

the oxidative stress induced or cytokine induced cell

death pathway. It is known that STZ injections cause

extensive necrosis in islets of Parp + / + mice while

the extent of necrosis was markedly lower in Parp- / -

islets43. It has also been shown that inhibition of

Parp-1 by synthetic inhibitors like 3-aminobenzamide

resulted in protection against necrosis. A potent

inhibitor of Parp, 5-iodo-6amino-1, 2 benzopyrone

(INH2BP), was found to protect rat islets and beta

cell line RIN-5F from cytokine induced damage44.

Fig. 2. Mechanisms of islet cell death. Flow chart depicts apoptotic and necrotic beta cell death cascades along with possible modes

of intervention. Causes/agents of beta cell death are indicated in red while agents/strategies for prevention of beta cell damage are

indicated in blue. Red arrows stand for possible sites of intervention. IL-11: Interleukin 11, IKK: Inhibitor of kappa kinase, iNOS:

inducible nitric oxide synthase, NO, Nitric oxide, STZ: Streptozotocin, ROS: Reactive oxygen species, PARP: Poly (ADP-Ribose)

Polymerase, CAT: Catalase: SOD, superoxide dismutase; GSH: glutathione peroxidase, NF-kB: Nuclear Factor kappa B. This figure

has been compiled and constructed by authors by referring to data cited in references 34-38.




NF-B activation is an important event in

inflammation, cellular death signaling and is often

activated by oxidative stress45. One of the key steps

in activation of the NF-B pathway is thestimulation of the I-B kinases46. Inhibition of NF-

B activation could be an effective means of

controlling islet cell death in vitro. Studies have

shown that IL-11, a regulatory cytokine, has been

effective in inhibiting NF-B activation in islets

leading to prevention of multiple low doses STZ

(MLD- STZ) induced diabetes in vivo47. Rehman

et al48 have demonstrated that adenoviral gene

transfer of the NF-B inhibitor I-B to isolated

human islets resulted in protection from IL-1

mediated dysfunction and apoptosis. Mouse islets

when transduced in s i tu by infusion of the

transduction peptide prior to isolation lead to 40

per cent of peptide transduction of cells. Delivery

of the IKK inhibitor transduction fusion peptide

(PTD-5-NBD) in situ to mouse islets resulted in

improved is let function and viabil i ty af ter

isolation48. Other studies involving rhIL-1 have

suggested a cytoprotective role of the recombinant

cytokine against alloxan induced toxicity in

diabetic rat islets49.

Apart from exogenously induced islet damage,

islets also suffer from damage due to endogenous

hyperglycaemia in vivo. It is known that high glucose

concentration causes apoptosis in cultured human

pancreatic islets of Langerhans. Data suggest that in

human islets, high glucose modulates the balance of

pro- and anti-apoptotic Bcl proteins towards cell

apoptosis50. Hyperglycaemia also causes production

of IL-1 by islet cells leading to cytotoxicity in

human pancreatic islets51. Chronic exposure to free

fatty acids alone or with hyperglycaemic conditions

lead to pancreatic cell death possibly employing

oxidative stress as the mechanism for cell

destruction52,53. Hence, blockage of multiple

pathways, rather than a single pathway, leading to

cell death may be necessary to fully protect cells

from destruction in vitro54. It is relatively easy to

study mechanisms of cell death, in its intricate

detail, along with modes of preventing the same and

its effect on diabetogenesis employing isolated islets

rather than animal experimentation.

Isolated islets for transplantation

Apart from studying strategies for prevention or

abrogation of cell death, isolated islets are widely

used for transplantation. Extensive studies have been

conducted wherein islets were transplanted into a

hyperglycaemic host and then checked for reduction

in hyperglycaemia. Till date it remains the most

successful means of achieving normoglycaemia in

humans55. Allo- or xeno- tranplantation of whole

pancreas is possible56, but it requires major surgery,

hence transplantation of islets57,58 or insulin producing

beta cells59,60 would be a more viable option.

Any successful transplantation depends on three

things, viz:

(i) Primary non function: Primary nonfunctioning

(PNF) of islets accounts for the bulk of graft losses.

Macrophages, the main effector cells in PNF,

release proinflammatory cytokines like IL-1 and

TNF-, which in turn recruit free radicals to mediatea nonspecific inflammatory response61. Lee et al62

have shown that the blockade of monocyte chemo-

attractant protein-1 (MCP-1) binding to CCR2, in

conjunction with sub therapeutic

immunosuppression, leads to islet allograft survival.

Hence, interruption of the leukocyte recruitment

through chemokine receptor targeting may be of

therapeutic benefit. Transfection of cytoprotective

genes to isolated pancreatic islets may contribute

to enhanced survival in transplant settings, e.g., the

overexpression of erythropoietin gene protects islets

from destruction and does not compromise islet

functionality63. It has been shown by Riachy et al64

using cell lines and human islets, that 1, 25-(OH)

2D3, the active metabolite of vitamin D3, was able

to induce and maintain high levels of A20, an anti-

apoptotic protein known to block NF-B activation,

thus promoting islet cell survival by modulating the

effects of inflammatory cytokines, which contribute

to cell demise64. Disruption of islet extracellular




matrice during pancreatic digestion leads to

induction of apoptotic pathways, thus increasing

cumulative PNF. Targeting the apoptotic pathway

by adenovirus-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective

effect on isolated islets. This was experimentally

proved with isolated macaque pancreatic islets. Bcl-

2 transfection ex vivo protected these islets from

apoptosis65.

(ii) Abrogation of immune rejection of graft and

recurrence of autoimmunity: The next hurdle, graft

rejection, can be dealt by the administration of

immunosuppressive drugs in the host, and/or

immunoisolation of the graft, or immunomodulation

of the graft or host or both 66 . Immuno-

compromisation enables allogenic and xenogenic

islet transplantation in preclinical, non human

primate models 67. Isolated islets are known to

reverse diabetes in immunocompromised nude mice

rendered diabetic by STZ. Although attractive, an

immunosuppression regime leaves the host

susceptible to other infections68, and these drugs

have adverse effects on insulin secretion by

cells 22,23,58 . A better alternative toimmunocompromization is to make the host tolerant

to the graft. Many different strategies have been

developed to achieve transplantation tolerance. In

one approach used by Oluwole et al 69 the

intravenous administration of genetically

engineered host dendritic cells (DCs) expressing

allo-MHC peptides, along with transient ALS

immunosuppression, resulted in induction of graft

tolerance. Similarly, induction of mixed chimerism

via bone marrow (BM) cells transplantation from

normal donors into autoimmune non obese diabetic

(NOD) mice has been shown to reverse insulitis and

prevented the development of diabetes and induces

tolerance to donor islet cells 70-74. This approach

however leaves the host susceptible to the graft

versus host disease (GVHD). Like this, Liang et al75

have described a radiation free regimen for

induction of chimerism, donor-specific tolerance,

reversal of insulitis, and resistance to diabetes

development in NOD mice model.

It is understood that complete T cell activation

requires two signals; T cell receptor (TCR)

interaction with peptide- MHC complex presented

by antigen presenting cells (APCs). This signal mustthen combine with another co-stimulatory signal,

mediated by interaction between distinct cell surface

molecules of APCs and T cells76. In absence of co-

stimulation, T cells undergo anergy and become non-

responsive. The B7/CD28/CTLA4 co-stimulatory

pathway plays a critical role in the regulation of T-

cell activation transplant rejection and autoimmunity.

Adams et al77 have used LEA29Y (BMS-224818), a

mutant of CTLA4-Ig along with repamycin and IL-

2R to effectively prevent the rejection of islet

allograft in a preclinical primate model.

Administration of co-stimulatory blockade (anti-

CD40L) has been reported to induce mixed

chimerism in NOD mice78,79. Pearson et al80 have

reported that an allelic variant of Idd3 gene is

responsible for prolonged islet allograft survival by

co-stimulatory blockade in NOD mice. MHC

antigens, expressed on APC of donor tissue, stimulate

a higher T cells response as compared to host APCs.

As passenger (donor) APCs are largely responsible

for co-stimulatory activity, graft immunomodulatorystrategies aim at depleting them from the islet grafts81.

These strategies include in vitro culture of graft at

suboptimal temperature for extended time82,83, UV-

B irradiation84, cryopreservation85,86 mitomycin C

treatment87, ICAM-1 specific monoclonal antibody

treatment88, co-transplantation of islets with sertoli

cells89 etc.

Xenotransplantation: The existing shortage of donor

islets makes it necessary to pool islets from different

donors or look for alternative islet sources. Foetal,

neonatal and adult porcine islets along with bovine,

murine, canine, avian and piscean islets have been

tested for this purpose and porcine islets have been

found to be an acceptable source for alternative islets.

It was observed that neonatal porcine pancreatic cell

clusters (NPCCs) contain mature endocrine cells and

bring about sustained normalization of blood glucose

levels when transplanted into kidney capsule of

diabetic nude mice. Rapid return to diabetic state was




observed after removal of islet grafts90,91. Recently,

Garkavenko et al92 have reported a follow up study

in human diabetic patients receiving porcine islets

for 9 yr. The finding that none of the patientsdeveloped viral infection hold promises for

xenotransplantation. Similarly, canine, bovine and

porcine islets have been successfully used for

xenotransplantation in a diffusion based bio hybrid

artificial pancreas93,94. Chick embryo pancreatic

transplants have shown reversal in experimental

diabetes of rats without immunosuppression 95.

Transient reversal of experimental diabetes in mice

has also been reported by transplantation

of chicken pancreatic islets96. Xenotransplantation of

fish islets into the non-cryptorchid testis has also

been carried out. Cryopreservation of principal islets

of teleost fish and their xenotransplantation has also

been studied97,98. Immunoisolation of islets by micro-

encapsulation is of great clinical potential in the

treatment of diabetes with xenotransplantation of

islets99-102. Lim and Sun, first described the alginate

micro-encapsulation of islets 10 2. Since then

immunoisolation has been regarded as the

technological key to xenotransplantation without

immunosuppression. The encapsulated isletscan be transplanted in, and retrieved from, the

peritoneal cavity with minimal invasive surgery.

Immunoisolation has facilitated the transplantation,

and consequent reversal of hyperglycaemia, from rat

to mice103,104, monkey to rats105, porcine tomurine, dog

to mice106, and even across a large species barrier

i.e., from rabbit to cynomolgus monkey107. An ideal

membrane for immunoisolation should be

biocompatible, non immunogenic, non cytotoxic,

differentially permeable to glucose, insulin, oxygen

and other growth factors required for prolonged

survival of graft and impermeable to

immunoglobulins, immune effector cells and their

recruiting cytokines108. Various natural biopolymers

like and synthetic materials have been used

extensively as immunoisolation material. We have

tested cellulose macrocapsules and molecular

dialysis membrane109,110, polyurethan111, chitosan-

PVP112 and chitosan-alginate113,114 microcapsules for

islet storage and transplantation purposes. Polymeric

biomaterials such as alginate115, agarose116, polyamide

4-6 membranes11 7, poly (ethylene glycol)

diacrylate118, polyvinylchloride acrylic copolymer119,

AN69120, polyurethane-polydimethylsiloxane121, poly(2-hydroxyethylmetacrylate) 122 have also been

proposed as immunobarriers.

Despite these successes the hurdles to be

conquered are monumentous. Theoretically it would

be best if an individual could simply regenerate its

own pancreas/islets.

cell replication and/or regeneration

Pancreatic islet cell growth can be mediated

by two separate mechanisms123. Either new islets can

generate from budding of the pancreatic ductal

epithelium 124-128 or from intra islet precursor

cells129,130, i.e., islet-neogenesis and by replication of

existing islet beta cells123,131 (Fig. 3).

Is let neogenesis : Neogenesis of islets primarily

occurs during foetal and perinatal stages of

development132, but has also been observed in the

regenerating adult pancreas133,134. In a population of well differentiated adult pancreatic islet cells, the

number of cells actually undergoing cell division

is small, measured to be between 0.5 to 2 per cent135.

Although the growth potential of the pancreatic islet

cells is limited, glucose (nutrients), c-AMP, and

certain polypeptide growth factors have been

reported to exert modest stimulatory effects on cell

growth and replication136 . Several authors have

reported differential effects of various growth factors

on islet neogenesis phenomenon. Movassat et al137,

have investigated the effects of keratinocytes growth

factor (KGF), in vitro, on cell differentiation from

undifferentiated pancreatic precursor cells. However

KGF does not help in cell replication137. Similarly

vascular endothelial growth factor (VEGF- ligand of

foetal liver kinase-1), has been shown to play a role

in the development of foetal rat islet-like structures

in vitro, possibly by stimulating the maturation of

endocrine precursor cells in the pancreatic ductal

epithelium138. Epidermal growth factor (EGF), an




activator of the MAP kinase pathway, increases the

mass of pancreatic epithelial cells but the absolute

number of developing endocrine cells decreases. On

the other hand, inhibition of MAPK pathway byPD98059 in the precursor cells leads to decreased

proliferation of epithelial cells but endocrine cell

differentiation was activated. Hence, MAPK pathway

determines the final mass of developing endocrine

tissue139.

cell replication: Sjoholm et al140, reported that

lithium treatment stimulates rat cell replication

and long term insulin secretion in vitro. The

relationship between cell replication and insulin

release was further investigated using neonatal rat

pancreatic monolayer cell cultures and the study

demonstrates the importance of glucose utilization

for both of these cell processes141. In another study

authors have demonstrated that even after complete

destruction of cells by STZ treatment in vitro,

foetal pancreatic cells retain the ability to regenerate cells142. The potential for large scale production

of endocrine tissue in vitro has been indicated,

however, more investigation needs to be carried out

on the various signals and pathways involved in

pancreatic development. An attempt to transduce

NPI (neonatal pancreatic islet) with gene of interest

i.e., PDX-1, allowed researchers to determine the

effects on islet maturation. The authors believed that

these transduced NPIs provide an effective tool to

study islet growth and maturation143. Transfection

of cells with tyrosine kinase receptors144 and

human islets with chimeric signaling receptors145

leads to ligand dependent cell proliferation. This

Fig. 3. Islet beta cell birth. Figure depicts different ways of islet beta cell neogenesis. Inset 1 to 4 depicts various stages in islet

generation from pancreatic duct epithelium stem cell monolayer (1), intense zone of activity (2), islet formation (3) and fully formed

mature islet (4). Further inset sequence represents trans-differentiation of islets from non-pancreatic stem cells viz. bone marrow,

acinar cells and hepatic progenitor cells. Insets 5: monolayer, 6: islet-like cell clusters and 7: Budding and maturation of islets.




strategy has potential to reduce the quantity of

human islets required for treatment of patients with

type 1 diabetes.

Conclusion

It is apparent that isolated islets form a handy

model system due to ease of isolation and

maintenance. Being miniature organ systems, they

do not require a nervous control and manipulations

like transfection studies related to signaling

pathways, insulin stimulation and secretion assays

are easy to perform in an in vitro system. Along with

ease of handling, studies based on isolated islets can

be extrapolated and data corroborate with related in

vivo findings, with high efficiency, thus supporting

the 3R principle of ‘Reduction, Refinement and

Replacement’ of animals in biomedical research.

These factors have led to islets being popularly used

as a compatible model system for diabetes and related

research. All above mentioned approaches are

considered in context of their current status, progress,

future challenges or limitations, and long-term

prospects for a cure. Although definitive success is

still at the horizon, the advances reviewed herepredict the future to be bright.

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Isolated Islets in Diabetes Research

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