Genes involved in pancreatic islet cell rejuvenation

Pancreas plays an important role in maintaining the glucose homeostasis. The deterioration of β-cells in the pancreas is a crucial factor in the progression of diabetes mellitus; therefore, the restoration of β-cell mass and its function is of vital importance for effective therapeutic strategies. The precise mechanism for increase in functional β-cell mass is still unknown. This review focuses on the importance of certain genes which are involved in the rejuvenation of pancreas. These genes are divided according to their functions into three categories: participate either in proliferation (mitotic division of differentiated β-cells), neogenesis/transdifferentiation (development from precursor cells) or inhibition of β-cell apoptosis (programmed cell death). The rate of β-cell rejuvenation is the balance among the rates of β-cell proliferation, neogenesis and apoptosis. Understanding these genes and their pathways may lead to the discovery of new drugs, target based gene delivery and development of safer antidiabetic drugs.


Introduction
Diabetes is a major cause of health concern in the world and is growing in epidemic proportions. It is assumed that in the next ten years it will become number one disease of the world 1 . Type-1 diabetes mellitus (T1DM) is an autoimmune disease while type 2 is mostly a lifestyle disease. Majority of people suffer mainly due to type-2 diabetes and is responsible for the current diabetes explosion. The detection early markers for the disease and its prevention is an active area of research to develop target based novel drugs.

Dysfunctional pancreas in diabetes
Insulin, a key polypeptide hormone secreted by the pancreas, targets several tissues for the utilization of glucose and thus maintains the glucose homeostasis.
Type 2 diabetes mellitus (T2DM) develops from a combination of genetic and acquired factors (such as changes in metabolic homeostasis) that impair β-cell function on one side, and tissue insulin sensitivity on the other 2,3 . Normally, β-cell mass can adapt to changes in metabolic homeostasis. Recurrence of these changes in metabolism creates a stress on pancreas often predating the on-set of T2DM by many years. This pancreatic stress causes β-cell mass expansion, through enhanced proliferation and neogenesis. The progression from this stress condition to a state of diabetes is inevitably associated with a decrease in the β-cell mass 2-4 . This β-cell loss arises due to an increase in β-cell apoptosis, which clearly outweighs replication and neogenesis.
The war against diabetes through the development of new drugs is an ongoing continuous process 5 . With Table I. Genes involved in beta cell proliferation

Genes proteins Functions References
Reg gene family (RegI, II, IIIα, IIIβ, IIIγ) Increase islets cell size and density, Regeneration of pancreas 16,22,24,25,27 Sox9 Stimulates proliferation and survival of pluripotent progenitors. 28 Hnf - Figure) also contributes for regeneration of pancreas in which gene/gene products play in conjunction either with (1) or (2). All these mechanism may contribute synergistically for the regeneration of pancreas. See Tables 1, 2 and 3 for gene/gene products involved in the above processes for pancreatic rejuvenation. Reproduced with permission from Nature Publishing Group, London, UK 6 .
the technological advancement, efforts are being made to rejuvenate the pancreatic cells or create artificial pancreas. Pancreatic rejuvenation can happen either due to proliferation of existing β-cells or differentiation of progenitor cells to β-cells 6 (figure), or due to decrease in β-cell apoptosis.

β-Cell proliferation
Islets regeneration refers to an increase in β-cell mass by proliferation and replication of existing islet cells. Several mouse studies 7-9 here shown that β-cells do not proliferate, however, lineage tracing studies [10][11][12][13][14] have confirmed that human β-cells proliferate and give rise to a population of progenitor/stem cell. Various genes and transcription factors are involved in this process viz. Reg (Regenerating islets derived proteins), Sox9, Hnf-6, NeuroD1, Neurogenin-3 and Netrin-1 (Table I). Besides these genes, certain peptides or their analogues such as glucagon like peptide-1/exendin-4 are also involved in islet regeneration. These observations are confirmed by using dipeptidyl peptidase (DPP) IV inhibitor sitagliptin in mice 15 .
So far, five REG proteins have been reported in humans that belong to Reg gene family. Some of the members of this family have been implicated in β-cell replication and/or neogenesis as shown in in vivo studies using transgenic and knockout mice 16 . These also preserve the β-cell mass in autoimmune type 1 diabetes 17 . This Reg family of genes are expressed in both young and old mice that were subjected to partial pancreatectomy 18 . In isolated rat islets, Reg1 mRNA levels were significantly increased by glucose, amino acids, foetal serum or specific growth factors such as insulin, growth hormone and platelet-derived growth factors (PDGF) 19 . PDGF receptor signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islet cells 20 . Disruption of RegI gene resulted in a significantly decreased rate of DNA synthesis and diminished β-cell hyperplasia in response to obesity, confirming the role of endogenous RegI in islets cell growth 21 . A study conducted by Huszarik et al 22 showed upregulation of RegII during diabetogenic process and also after adjuvant therapy in NOD mice. While all Reg family mRNAs can be detected from total pancreas, RegII and RegIIIα genes have been detected in pancreatic islet cells as confirmed by immunofluorescence 23 and RegIIIα expression was remarkably increased during pregnancy in rats 24 . Mice overexpressing RegIII β was resistant to streptozotocin induced diabetes mellitus 25 . RegIIIγ, another member of Reg family of genes is also found to be involved in regeneration of pancreas. REG III protein was found to be expressed only in regenerating islets and not in normal rat pancreas 26 and its gene expression level induced 10-100 folds on day 3 of pancreatectomy 27 . These data suggest that there is a strong link between Reg gene family and rejuvenation of pancreatic islets.

Transcription factors in β-cell proliferation
Certain transcription factors (Sox9, Hnf-6, Ngn-3 and NeuroD1) are also found to be involved in the proliferation of β-cells. SOX9 is the first specific marker and maintenance factor of multi-potential progenitors during pancreatic organogenesis. SOX9, in the embryonic pancreas stimulates proliferation and prevents apoptosis of pluripotent progenitor cells. It controls pancreatic progenitor cell maintenance by modulating Notch signal transduction. The phenotypic alterations in the Sox9-deficient pancreas shows a striking resemblance to the pancreatic defects associated with mutations in components of the Notch signalling pathway, thus establishing a possible link between Sox9 and the notch signal transduction pathway for stem cell maintenance 28 . The hepatocyte nuclear factor 6 (Hnf-6), homeodomain-containing transcription factor, is an important regulator of endocrine development. HNF6 is expressed in early pancreatogenesis in all endodermally derived cells, but is not detected in differentiated endocrine cells at late-gestation 29 . Hnf-6 null mice embryos showed impaired endocrine differentiation and perturbed duct morphogenesis during embryogenesis 30 . In addition to defects in endocrine development, Hnf-6 null embryos showed defects in duct development 31 . Loss of Hnf-6 from Ngn-3 expressing cells did not affect β-cell function or glucose homeostasis suggesting that Hnf-6 is dispensable for later events of endocrine differentiation. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn-3 expression and proper pancreatic duct morphology 32 . NeuroD1, a downstream target of Ngn-3, carries on the endocrine differentiation programme initiated by Ngn3 and participates in the maintenance of the differentiated phenotype of the mature islet cells 33 . During pancreatic endocrine development, Ngn-3 acts early to determine endocrine cell fate, while NeuroD1 directs endocrine cell differentiation 34 . At early stage of life, mice lacking a functional NeuroD1 (also called as BETA2) gene exhibit a striking reduction in the number of insulinproducing β-cells and failed to develop mature islets with a marked hyperglycaemia. Attempts to rescue the diabetic phenotype by administration of insulin were unsuccessful, suggesting that the mutant animals were unable to respond to insulin, have become insulin resistant, or perhaps contained additional defects 35 . Thus BETA-2 is required for the expansion of the pancreatic β-cells population, as well as other islet cell types which are involved in the development of endocrine cells into islets of Langerhans 34 .
Netrins are laminin-like diffusible chemotactic proteins involved in pancreatic morphogenesis and play a role in the regulation of duct-cell and foetal islet cell migration. In adult rat pancreas, Netrin-1 mRNA was practically undetectable. After duct ligation, its expression was very low in the head part of the pancreas whereas it was strongly upregulated in the tail part at 3 rd , 5 th and 7 th day of post-ligation with the maximum expression on day 5 36 . Netrin-1 mRNA was found to be expressed by islet cells and exocrine cells with ductal characteristics. These observations suggest that Netrin-1 plays a role in pancreatic morphogenesis, both prenatally and in the regenerating adult rat pancreas.

Transdifferentiation of pancreas
Islet neogenesis specifically refers to an increase in β-cell mass via transdifferentiation of adult pancreatic stem cells, putatively found in the ductal epithelium or acinar tissue. Trans-differentiation involves in the conversion of alpha or delta cells of the pancreas into insulin producing β-cells. Various genes/proteins contribute to this process. These include INGAP, Gastrin,MafA,Foxa2,Nkx2.2,Nkx6.1,Pax4,etc. (Table II).
INGAP (islets neogenesis associated protein) is a member of the C-lectin protein family that serves as the initiator of a cascade of events that culminates in islet neogenesis and can reverse diabetes in streptozotocininduced diabetic C57BL/6J mice 37 . These studies were further confirmed in beagle dogs 38 . There was also a significant increase in insulin gene expression in the INGAP treated animals. INGAP is also found in human pancreas during pathological states involving islet neogenesis which further suggests that INGAP is of primary importance in the process of islet neogenesis 39 . Gastrin, a classical gut hormone secreted by G cells in the stomach lining is found to stimulate pancreatic β-cell neogenesis. Intravenous infusion of gastrin into the ligated duct cells, resulted in a doubling of the β-cell mass in rats 40 , due to high expression of gastrin/ cholecystokinin (CCK) B receptors in duct ligated cells 41 . These observations were confirmed using EGF plus gastrin combinatorial therapy, which showed an increase in insulin positive cells in human islet 42 . In another study using GLP-1 and gastrin in NOD mice, there was a significant reduction in blood glucose due to increase in pancreatic β-cell mass and insulin content 43 . MafA a member of Maf subfamily of leucine zippers is capable of strongly activating the insulin promoter. Maf factors play important roles in cellular differentiation of exocrine cells to β-cells 44 . MafA is restricted to β-cells and known to be important in the embryonic development of pancreas 45 . It has been observed that MafA expression is decreased during the diabetic condition 46 .

Transcription factors in pancreatic neogenesis
There are several transcription factors involved both in neogenesis and replication. However, it is not clear whether these work alone or in combination with other transcription factors in a coordinated manner. The list of the transcription factors and their role in transdifferentiation is summarized in Table II. Pancreatic duodenal homeobox-1 (Pdx-1), a homeobox transcription factor, besides being involved as a regulator of pancreatic development (the differentiation and gene expression in the β-cell) 47 , Pdx1 also turns out to be a major player in the maintenance of an adequate pool of healthy β-cells in adults 48,49 . It maintains the homeostasis between β-cell neogenesis and apoptosis. In mice with a 50 per cent reduction in Pdx1, the isolated islets showed more susceptibility to apoptosis at basal glucose concentrations along with impaired ability to maintain β-cell mass with age. Its expression is shown to be down-regulated during hyperglycaemic condition 50 . The survival functions of Pdx1 may be mediated by insulin/IGF signaling acting through the forkhead transcription factor Foxo1 (forkhead/winged helix transcription factor). Foxa2 (formerly known as Hnf-3) is a key regulator of foregut development that plays an essential role in the cell type-specific transcription of the Pdx-1 gene in the pancreas 51 . On deletion of Foxa2 in mice, there is a significant downregulation of Pdx-1 mRNA 52 . This shows that  Gastrin Induces islet β-cell neogenesis from pancreatic exocrine duct cells. 40

Nkx6.1
Maintaining & expanding population of β-cell precursors as these progress from precursors to differentiated β-cell.

Pax4
Expressed in endocrine cell progenitors and directs formation of β and delta cells.
is an essential upstream factor that regulates Pdx-1 mRNA levels in β-cells.
Nkx2.2 is another essential pancreatic transcription factor that affects the expression of ghrelin during pancreatic development 53 . Nkx2.2-null mice lose all β-cells and the majority of the α-cells; and the islet is predominantly populated by gherlin expressing cells. The discovery that ghrelin cells often replace the other endocrine populations in the pancreas 54 suggests a lineage relationship between the ghrelin producing epsilon cells and other hormone-producing populations 55 . In another study, disruption of the Nkx2.2 gene has shown to lead to the accumulation of incomplete differentiated β-cells that express some β-cell markers, but not insulin 56 . This illustrates the role of Nkx2.2 in pancreatic endocrine cell differentiation. The phenotypic effects of Nkx2.2 mutant mice may in part result from the loss of other homeodomain transcription factor Nkx6.1. In pancreas, Nkx6.1 also follows an expression pattern similar to that of Nkx2.2; but restricted to the β-cells alone 57 . Deletion of the Nkx6.1 gene in mice caused a marked reduction of β-cells. In the same study, with the double knockout animal model (Nkx2.2/Nkx6.1) showed the same phenotype as of Nkx2.2 single mutant 58 . This shows that Nkx6.1 functions downstream of Nkx2.2 in pancreatic development. The paired homeobox transcription factor Pax4 appears to be a strong candidate for specifying β-cell lineages. Mice deficient in Pax4 fail to develop beta and delta cells within the pancreas suggesting that Pax4 expression commits selected endocrine precursors towards the beta and delta cell lineage 59 . The Pax4 genes are expressed during the embryonic pancreatic development, but later on these are restricted to β-cells alone. Inactivation of Pax4 in mice results in the improper maturation of alpha-and β-cells 60 . This indicates the role of PAX4 in the maintenance of progenitors and in maturation of β-cells.

Inhibition of β-cell apoptosis
Under normal circumstances, apoptosis is highly regulated to maintain normal physiological function of the cells. In diabetes, during excess stress, the pancreatic cells not only undergo apoptosis but also become necrotic and are unable to secrete insulin. A study conducted by Butler et al 61 indicated that increased apoptosis rather than decreased neogenesis/ proliferation might be the main mechanism leading to reduced β-cell mass in T2DM. Thus, decrease in the rate of apoptosis itself, may increase the β-cell mass via proliferation. Several genes/proteins are involved in pancreatic apoptosis and their functions are summarized in Table III. Perforin (pore forming protein) is a cytolytic protein, initiating apoptosis by inducing minimal cell membrane damage while effectively releasing Table III. Genes involved in inhibition of beta cell apoptosis

Pdx-1
Granzymes Possesses anti-apoptotic activity that helps facilitate the maintenance of β-cell mass.
A serine protease, that activates Bid (a pro-apoptotic factor) essential for death-receptor induced apoptosis of islets. Thioredoxin-interacting protein (TXNIP) It is a pro-apoptotic factor that plays an essential role in glucose toxicity-induced β-cell apoptosis.

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granzymes from the endosomal compartment into the cytosol 62 . Of the granzyme family, granzyme A and B are the most common in human and mouse 63 . Granzyme A induces single-strand DNA breaks, while granzyme B cleaves specific substrates including caspases and the pro-apoptotic molecule Bid 64 . Activated Bid is targeted to the mitochondria where it sequesters antiapoptotic members of the Bcl-2 family, allowing the oligomerization of Bax and/or Bak which mediates loss of mitochondrial outer membrane potential, release of cytochrome C and causes irreversible apoptosis 65 . During hyperglycaemic condition its expression is elevated in β-cells, thus increasing the rate of apoptosis 51 . Deficiency in Bid prevents β-cells from undergoing mitochondrial apoptotic pathway 66,67 . Caspase-3 has been extensively studied in various tissues due to its role as the principal executioner of apoptosis 68 . As such, Caspase-3 is an attractive target to inhibit apoptosis in diseased conditions including diabetes 69 . Caspase-3 null (Casp3 _ / _ ) mice were found to be protected from developing diabetes in a multiplelow-dose streptozotocin autoimmune diabetic model 70 . This illustrates the importance of Caspase-3 in β-cell death and its activity is found to be increased during the diabetic conditions. An attractive regulator of β-cell replication and survival after birth is Survivin. This protein blocks the functions of Caspases in the mitochondria-dependent cell death pathway, protecting cells from apoptosis 71 . Deletion of Survivin within the mice endocrine pancreas results in diabetes manifested by hyperglycaemia and polyuria. Exogenous expression of Survivin in streptozotocin -induced diabetic model protects the β-cells from apoptosis 72 . Thus, it may play a role in the replication and/or survival of matured β-cells 73 . Surprisingly it is modestly upregulated in diabetic patients 74 which may help to assuage diabetes.
Beside survivin, there are other proteins present in pancreas which prevent apoptosis. BCL-xL, an anti-apoptotic protein coded by the "survival gene", is involved in the inhibition of apoptosis. Marked overexpression of Bcl-xL, resulted in a severe defect in insulin secretion and hyperglycaemia in transgenic mice 75 . Under conditions of stress, β-cells require BCL-xL to maintain their survival in vivo 48 . Myc is a potent inducer of both β-cell proliferation and apoptosis in vitro 76 . Myc sensitizes cells to a variety of apoptotic triggers rather than directly inducing apoptosis by itself 77 . Sustained Myc activation leads to initial hyperplasia and increased apoptosis later on, suggesting that apoptosis ultimately predominates over proliferation 78 . In chronic hyperglycemia, an increase in the expression of Myc was observed in pancreas 79 . A thioredoxin-interacting protein (TXNIP), a regulatory protein, is involved in the inhibition of thioredoxin and thereby modulates the cellular redox state and promotes oxidative stress 80 . Its overexpression in β-cells is found to induce apoptosis 81 , and the process involves the activation of intrinsic mitochondrial pathway, while the Endoplasmic reticulum (ER)mediated cell death remains unaffected. It was demonstrated that their mRNA expression levels are elevated during the diabetic conditions 82 . In a recent study, huntingtin-interacting protein  is found to posses anti-apoptotic property of β-cells. This also plays an important role in glucose-stimulated insulin secretion 83 . Bace2 (Beta site amyloid precursor protein cleaving enzyme 2), a proteolytic enzyme acts as a negative regulator of β-cell mass via inhibiting Tmem27 expression. Overexpression of Tmem27 or ablation of Bace2 leads to an increased β-cell mass by inhibiting apoptosis 84 .

Future prespective
Several lifestyle diseases such as T2DM, obesity are increasing significantly. It has been predicted that in near future diabetes will overtake all the current infectious and non-infectious diseases. In spite of rapid progress in our understanding regarding the pathophysiology of diabetes, challenges remain high due to increased complexity of the disease contributed by genetic and environmental factors. In future, there need to be more emphasis on the prognosis and better treatment for diabetes associated diseases, and on the discovery of reliable biomarker(s) for its early detection. Development of new technologies i.e. rejuvenation of the pancreas by small molecules or gene targeted drug delivery, pancreatic transplantation, stem cell therapy and creating artificial pancreas, etc. will be the mainstay of the diabetes research. Thus, it becomes essential to have better understanding of the various pathways at the molecular level that are involved in pancreatic islet cell rejuvenation.