Surg Today DOI 10.1007/s00595-015-1215-2
REVIEW ARTICLE
Pancreatic regeneration: basic research and gene regulation Kenji Okita1 · Toru Mizuguchi1 · Ota Shigenori1 · Masayuki Ishii1 · Toshihiko Nishidate1 · Tomomi Ueki1 · Makoto Meguro1 · Yasutoshi Kimura1 · Naoki Tanimizu3 · Norihisa Ichinohe3 · Toshihiko Torigoe2 · Takashi Kojima4 · Toshihiro Mitaka3 · Noriyuki Sato2 · Norimasa Sawada5 · Koichi Hirata6
Received: 4 February 2015 / Accepted: 19 May 2015 © Springer Japan 2015
Abstract Pancreatic regeneration (PR) is an interesting phenomenon that could provide clues as to how the control of diabetes mellitus might be achieved. Due to the different regenerative abilities of the pancreas and liver, the molecular mechanism responsible for PR is largely unknown. In this review, we describe five representative murine models of PR and thirteen humoral mitogens that stimulate β-cell proliferation. We also describe pancreatic ontogenesis, including the molecular transcriptional differences between α-cells and β-cells. Furthermore, we review 14 murine models which carry defects in genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.
* Toru Mizuguchi [email protected] 1
Department of Surgery, Surgical Oncology, Sapporo Medical University, Sapporo, Hokkaido 060‑8543, Japan
2
Department of Pathology I, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
3
Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
4
Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
5
Department of Surgical Pathology II, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
6
Department of Surgery, JR Sapporo Hospital, N‑3, E‑1, Chuo‑Ku, Sapporo, Hokkaido 060‑0033, Japan
Keywords Pancreatic regeneration · Transcriptional factors · Gene regulation
Introduction Pancreatic regeneration (PR) is an interesting phenomenon which could provide clues as to how the control of diabetes mellitus might be achieved. The pancreatic functions can be divided into exocrine and endocrine functions; however, most research into the physiology and morphology of islet cells has focused on β-cells, which only perform endocrine functions. Despite the fact that liver regeneration has been extensively investigated [1–3], little is known about PR. The liver and pancreas both develop from the endoderm and share a lot of gene regulation mechanisms; however, they have markedly different regenerative abilities. In this review, we summarize the murine models of PR and review the humoral mitogens for β-cell proliferation and pancreatic ontogenesis. We also review the murine models that carry defects in the genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.
Models of pancreatic regeneration PR is known to occur in the neonatal period in humans, as well as in type I diabetes, obesity, and pregnancy. However, a comprehensive study of human PR after 50 % resection involving 13 patients who underwent two consecutive partial pancreatic resections did not obtain any evidence of β-cell regeneration during an observation period of 1.8 ± 1.2 years [4]. Thus, a high incidence of diabetes is probably inevitable after pancreatectomy. Although human
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PR could be totally different from the PR seen in other species, numerous animal models have been developed to investigate the molecular and physiological mechanisms of PR. Scow et al. tried to produce a rat model of total pancreatectomy to investigate the physiological mechanism responsible for the acute onset of diabetes. However, they found that it was difficult to perform total pancreatectomy in rats. In addition, the rats did not develop diabetes and remained alive and in good health for several months [5]. Anatomical peculiarities are the main reason why it is technically difficult to perform total pancreatectomy in rats. Although only a very small amount of tissue is left in the area between the duodenal wall and bile duct after total pancreatectomy, it is large enough to enable the rats to maintain glucose homeostasis. Due to these difficulties, Houry et al. reported a modified total pancreatectomy technique for rats which involved a liver transplant [6], and Migliorini et al. described a two-stage rat total pancreatectomy procedure [7]. Wenger et al. attempted to develop a rat model of surgically induced diabetes by making further modifications to the above surgical procedures because previous models exhibited incomplete diabetes after surgery [8]. Partial pancreatectomy has also been used to investigate β-cell physiology. The anatomical nomenclature for rat partial pancreatectomy was first described in 1964 [9]. Subsequently, Pearson et al. observed PR in rat models subjected to 52, 69, or 91 % pancreatectomy [10]. The 52 % model was considered to represent splenic resection and was renamed the 50 % model, the 69 % model was considered to represent gastrosplenic resection and was referred to as the 70 % model, and the 91 % model was considered to represent subtotal resection and was renamed the 90 % model. Partial pancreatectomy stimulated PR in each of these models. Specifically, it caused 21, 32, and 78 % increases in the pancreatic weight in the 50, 70, and 90 % models, respectively (Table 1). Pancreatic duct ligation (PDL) is another experimental model of PR. Although PDL has been extensively studied, it has not been conclusively determined whether it promotes PR with the involvement
of both exocrine and endocrine cells [11]. PDL has been reported to induce the development of putative pancreatic precursor cells, which express neurogenin 3 (Ngn3) [12], carbonic anhydrase II [13], and pancreas-specific transcription factor 1 subunit alpha (Ptf1a) [14]. However, in another study β-cell regeneration was not observed after PDL [11].
Humoral growth factors for β‑cells Several humoral growth factors have been shown to act as mitogens to stimulate β-cell proliferation. Betacellulin was discovered by Shing et al. in 1993. It is a member of the epidermal growth factor (EGF) family and exhibits 50 % homology with transforming growth factor (TGF)-α [15]. Betacellulin was identified as a 32-kilodalton (kDa) glycoprotein in conditioned medium derived from mouse pancreatic β-cell tumors. The injection of betacellulin ameliorated glucose intolerance in an alloxan-induced mouse model of diabetes by increasing β-cell volume [16]. Other members of the EGF family, including EGF itself [17], TGF-α [18], and keratinocyte growth factor [19], also act as β-cell mitogens. In addition, hepatocyte growth factor (HGF) has been shown to have strong post-injury mitogenic effects in multiple organs. Although it is uncertain whether the administration of HGF promotes β-cell proliferation, HGF has been shown to promote β-cell proliferation after partial pancreatectomy and streptozocin-based β-cell ablation [20]. Glucagon-like peptide-1 (GLP-1) and exendin-4, which are incretins, are also considered to β-cell mitogens [21]. These proteins have similar structures and bind to the GLP-1 receptor [22]. The glucose-dependent insulinotropic polypeptide is another incretin that promotes β-cell proliferation and controls glucose homeostasis [23]. Throughout the world, long-acting incretin analogs are now widely used in combination with inhibitors of the degrading enzyme dipeptidyl peptidase-4 to treat type II diabetes [24]. Gastrin can also stimulate β-cell neogenesis and the proliferation
Table 1 Murine pancreatectomy models Operation
Author
Years
Journal
Techniques
Result
Total pancreatectomy
Scow
1956
Endocrinology
Total pancreatectomy
No diabetes
Houry
1983
Eur Surg Res
Transplant technique Total splenopancreatectomy
Diabetes after 8 h
Migliorini
1970
Diabetes
Two-stage technique
Diabetes after 24 h
Wenger
1990
J Surg Res
Radical splenopancreatectomy
Diabetes
Pearson
1977
Gastroenterology
52 % (rounded off to 50 %): splenic resection
21 % increase in pancreatic weight
69 % (rounded off to 70 %): gastrosplenic resection
32 % increase in pancreatic weight
91 % (rounded off to 90 %): subtotal resection
78 % increase in pancreatic weight
Partial pancreatectomy
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Surg Today Table 2 Mitogens for b cell proliferation Group
Description
Betacellulin Epidermal growth factor Transforming growth factor–α Keratinocyte growth factor Growth factors Hepatocyte growth factor/c-met Incretins Glucagon-like peptide-1 Exendin-4 Glucose-dependent insulinotropic polypeptide
suggests the existence of other proteins with similar functions to betatrophin [35] (Table 2).
EGF family
Others
Gastrin Glucagon-like peptide/gastrin Epidermal growth factor/gastrin Islet neogenesis-associated protein pentadecapeptide Mangiferin Silymarin Betatrophin
of β-cell masses from transdifferentiated pancreatic exocrine cells [25]. In addition, the combined administration of gastrin and GLP-1 or EGF resulted in greater mitogenic activity in comparison to that observed with the administration of a single mitogen [26]. Islet neogenesis-associated protein pentadecapeptide (INGAP-PP) is a section of the INGAP protein (amino acids 104–118) that was discovered in 1992 [27], and which has been experimentally used in the treatment of type I and type II diabetes in a randomized double-blinded clinical study [28]. Although it is not yet commercially available for clinical use, it was shown to modestly improve the clinical parameters of the diabetes patients to whom it was administered [28]. Mangiferin is a natural xanthonoid that can be extracted from mangos and which is commercially available [29]. As well as acting as a β-cell mitogen, mangiferin possesses multiple other functions, such as anti-oxidant, insulin-stimulating, and antiviral activities [29]. Silymarin is a herbal medicine that is derived from Silybum marianum (milk thistle) [30, 31]. Seven flavonolignans, including silybin (which accounts for 50–70 % of the flavonolignans found in S. marianum), and the flavonoid taxifolin can be extracted from S. marianum [31]. Soluble biosynthesized silybin has been developed in order to increase the bioavailability of the molecule [31]. The administration of silymarin resulted in increased serum insulin levels and β-cell neogenesis in a partially pancreatectomized rat model [32]. Betatrophin (also known as ANGPTL8) is an angiopoietin-like protein that exhibits some functional overlap with ANGPTL3 [33]. Although betatrophin promotes the proliferation of pancreatic β-cells and increases insulin secretion [34], betatrophin knockout (KO) mice are born normally and develop lean bodies. This
Pancreatic ontogenesis The pancreas develops from the foregut endoderm. Its development is initially associated with the activation of pancreatic and duodenal homeobox 1 (Pdx1) [36, 37]. In addition, sonic hedgehog (Shh) determines the fate of duodenal/pancreatic precursor cells [38]. The turning off of Shh expression and the upregulation of Ptf1a expression are necessary for the differentiation of duodenal/pancreatic precursors into pancreatic precursors; otherwise, they follow an intestinal fate [36]. The further induction of Ptf1a results in the differentiation of pancreatic precursors into exocrine cells. On the other hand, the activation of Ngn3 encourages pancreatic precursors to become endocrine progenitors [39]. Human transcription factor 2 (TCF2; also known as vHNF1 or HNF1β) is a POU-homeobox transcription factor that represses Shh and activates Ptf1a [40]. TCF2 also activates Ngn3, islet-1 (Isl1), and Pax6 [40]. The turning off of hepatocyte nuclear factor 6 (HNF-6) and Ptf1a expression is required for the differentiation of pancreatic precursors into endocrine cells [36]. On the other hand, the musculoaponeurotic fibrosarcoma oncogene family (Maf)B induces the transformation of endocrine cells into α-cells and β-cells [41]. The aristaless-related homeobox (Arx) and paired box 4 (Pax4) genes mutually regulate each other’s expression and determine the final functions of endocrine cells. The induction of Arx is seen in α-cells and ε-cells. On the other hand, upregulated Pax4 expression is observed in β-cells and δ-cells. Furthermore, it was demonstrated that brain-4 (Brn4) and Iroquoisrelated homeobox gene 2 (Irx2) are specifically expressed in α-cells, whereas the Nirenberg and Kim homeobox (Nkx) genes (Nkx 2.2, Nkx 6.1, and MafA) are specifically expressed in β-cells (Fig. 1).
Mouse KO models of key transcription factors for pancreatic ontogenesis PDX1 KO mice Pdx1 (also known as IPF1, IDX-1, and STF-1) is a homeobox-like homeoprotein that plays a crucial role in pancreatic development [42]. The homozygous depletion of Pdx1 results in the complete absence of the pancreas and death within a few days of delivery [43]. Heterozygous depletion is not lethal, but causes diabetes and a reduction in the number of β-cells [44] (Table 3).
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Fig. 1 Pancreatic ontogenesis and transcriptional gene regulation. Pdx1 pancreatic and duodenal homeobox 1, Shh sonic hedgehog, Ptf1a pancreasspecific transcription factor 1 subunit alpha, TCF transcription factor, vHNF1 variant hepatocyte nuclear factor, HNF hepatocyte nuclear factor, Ngn3 neurogenin 3, Isl1 Islet-1, Maf Musculoaponeurotic fibrosarcoma oncogene family, Pax paired box, Nkx Nirenberg and Kim homeobox, Arx Aristalessrelated homeobox gene, Brn brain, Irx Iroquois-related homeobox gene, PP-cells pancreatic polypeptide-producing cells
Endoderm Pdx1 on Duodenal/Pancreatic precursors Duodenal mucosa
Shh on
Shh off Ptf1a on
TCF2 (vHNF1, HNF1β)
Pancreatic precursors Endocrine progenitors
Ptf1a on high
Exocrine cells
Endocrine cells
Ngn3 on Isl1 on HNF6 off Ptf1a off
MafB Arx on Pax6 on
α-cells glucagon
ε−cells ghrelin
Brn4 Irx2
Pax4 on Nkx2.2, 6.1 Pdx1 on high β-cells insulin
δ-cells somatostatin
γ-cells (PP-cells) pancreatic polypeptide
MafA
Table 3 Mouse knockout models of key transcription factors for pancreatic organogenesis Genetic defect
Morphology
Homozygous phenotype
Pdx1 Ptf1a (PTF1-p48) Sonic hedgehog (Shh)
Reduced insulin and glucagon production Pancreatic agenesis Severe lung branching defects Essential for coronary vascular development
No pancreas, death occurs within a few days Death occurs shortly after birth Death occurs during the embryo stage
TCF2/vHNF1
Defective visceral endoderm formation Pancreatic agenesis by embryonic day 13.5
Death occurs during the embryo stage
Ngn3
Normal at birth, but death occurs after 2–3 days due to dehydration
Death occurs 2–3 days after birth
Isl1 HNF6
The dorsal pancreatic mesenchyme does not form Reduction in the number of endocrine cells
MafB KO Pax4
Reduced numbers of α- and β-cells Increased production of glucagon and ghrelin Decreased numbers of β- and δ-cells
Death occurs during the embryo stage Growth retarded and 75 % die between 1 and 10 days after birth due to liver failure Death occurs at birth due to central sleep apnea Normal appearance, but die within 3 days
Arx
Increased numbers of β- and δ-cells No mature α-cells
Death occurs within 2 days after birth
Nkx2.2
No β-cells, but precursor cells survive Few α- or PP-cells
Death occurs shortly after birth
Nkx6.1 Pax6
No β-cells (including precursors) No α-cells, but other cell types are present
Death occurs immediately after birth Death occurs a few minutes after birth
MafA
Normal pancreas, but age-dependent functional defects
MafA deficiency does not confer embryonic lethality
Pdx1 pancreatic and duodenal homeobox 1, Ptf1a pancreas-specific transcription factor 1 subunit alpha, TCF transcription factor, vHNF variant hepatocyte nuclear factor, Ngn3 Neurogenin 3, Isl1 Islet-1, HNF hepatocyte nuclear factor, Maf musculoaponeurotic fibrosarcoma oncogene family, Pax paired box, Arx Aristaless-related homeobox gene, Nkx Nirenberg and Kim homeobox, PP-cells pancreatic polypeptide-producing cells
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Ptf1a (PTF1‑p48) Ptf1a (also known as PTF1-P48) is a 48-kDa class B basic helix–loop–helix (bHLH) protein that acts as the DNAbinding subunit of the trimeric transcription factor PTF1 [45, 46]. Ptf1a is a key regulator of the fate of pancreatic precursors (i.e., whether they differentiate into exocrine or endocrine cells) [46]. The null mutation of Ptf1a results in death shortly after birth [47]. The exocrine pancreas was absent from embryos carrying a null mutation in Ptf1a, although all four endocrine cell lineages were present [47]. However, endocrine cells were found in the spleen and functioned until postnatal death [47].
[61], as the dorsal pancreatic mesenchyme does not form properly [62]. HNF‑6 KO mice HNF-6 is a cut-like homeodomain protein and a key component of the pancreatic transcription factor cascade [63]. The main phenotypic features of HNF-6 KO mice are as follows: (I) the development of the endocrine pancreas is severely inhibited; (II) the appearance of the islets of Langerhans is delayed; and (III) when the islets form, they display a perturbed architecture and contain cells that have not reached full maturity [64].
Sonic hedgehog (Shh)
MafB KO mice
Shh is a mammalian hedgehog gene, which is essential for the development of numerous organs [48], and which has been reported to cause the formation of an annular pancreas [49]. Shh-deficiency is lethal and results in death shortly after birth [49, 50]. Shh is expressed in the primitive gut, including the presumptive pancreatic area [49], and is repressed by activin B. Shh inhibition is required to initiate the pancreatic differentiation program [51].
MafB is also a member of the basic-leucine zipper-containing transcription factor family and is distinguished from other large Maf molecules by the presence of an N-terminal transactivation domain [41, 65]. MafB-deficient mice die at birth due to central sleep apnea [65]. MafB is a regulatory transcription factor that plays a key role in the development of mature α- and β-cells [41]. Pax4 KO mice
TCF2/vHNF1 The human transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1β (a human POU-homeobox protein also known as vHNF) [40]. A mutation in the TCF2 gene is responsible for maturity-onset diabetes of the young type 5 (MODY5) [40, 52]. TCF2 plays a crucial role in controlling pancreatic development, and Ptf1a expression is not induced in the absence of TCF2 expression [40]. In addition, null mutation of the TCF2 gene causes pancreas agenesis and death before gastrulation [40]. Ngn3 KO mice Ngn3 is a member of a family of bHLH transcription factors that are expressed in the embryonic pancreas [53]. Ngn3 mutant mice are morphologically indistinguishable from wild-type mice and feed normally after birth, but die after 2–3 days [39]. This phenotype is reminiscent of the phenotype of diabetic mice with mutations in neurogenic differentiation (NeuroD)/BETA2 [54], Pdx1 [55], Nkx 2.2 [56, 57], or Pax4 [58, 59].
Pax4 is a unique member of the Pax family of transcription factors that is exclusively expressed during the embryonic stage [66]. Although heterozygous Pax4-deficient mice have normal lifespans and are fertile, homozygous mice die within 3 days due to dehydration [59]. Arx KO mice Defects in the Arx gene result in the opposite phenotype to that seen in Pax4-deficient animals [67]; i.e., a reduced number of α-cells, and greater numbers of β- and δ-cells. Direct mutually antagonistic transcriptional repression has been shown to occur between Arx and Pax4 [68]. Nkx2.2 KO mice Nkx2.2 is a member of the vertebrate homeodomain transcription factor gene family and is almost homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene [69]. Homozygous Nkx2.2 deficiency results in severe hyperglycemia and death shortly after birth [57]. Nkx2.2 is required for the final differentiation of pancreatic β-cells [57].
Islet‑1 KO mice Nkx6.1 and Nkx6.2 KO mice Isl1 is a transcriptional regulator of the LIM-homeodomain and plays a key role in the regulation of islet development [60]. Isl1 deficiency results in death during embryogenesis
Although the pancreas develops normally in Nkx6.2 singlemutant mice, Nkx6.1 mutants exhibit severe defects in their
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β-cells, including in their β-cell precursor cells [70]. In addition, Nkx6.1/Nkx6.2 double-mutant embryos display a markedly reduced number of α-cells [71]. Pax6 KO mice Pax6 is the gene responsible for human aniridia and mutant mice with small eyes [72]. Homozygous Pax6-deficient mice are born without eyes and do not develop distinct islets. In addition, they exhibit a marked deficiency of α-cells [73]. Musculoaponeurotic fibrosarcoma oncogene family A KO mice The musculoaponeurotic fibrosarcoma oncogene family (Maf)A is a basic-leucine zipper transcription factor that plays an important role in maintaining β-cell function [74]. In MafA KO mice, although the pancreas develops fully, age-dependent defects are observed in glucose homeostasis [74, 75].
Conclusion We reviewed PR, including its ontology and molecular biology, from a basic point of view. Knowledge on PR is accumulating rapidly, and the molecular regulatory mechanisms responsible for PR are being revealed. Various transcription factors are critical for pancreatic development and survival. In the future, we hope to create pancreatic cells for therapeutic purposes. Acknowledgments Part of this study was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No.24659592) to T. Mizuguchi, T. Torigoe, N. Sato, and K. Hirata. Part of this study was also supported by a Health Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare (No. 2601023) to T. Mizuguchi, T. Torigoe, K. Hirata, and N. Sato.
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64. Jacquemin P, Durviaux SM, Jensen J, Godfraind C, Gradwohl G, Guillemot F, et al. Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3. Mol Cell Biol. 2000;20:4445–54. 65. Blanchi B, Kelly LM, Viemari JC, Lafon I, Burnet H, Bevengut M, et al. MafB deficiency causes defective respiratory rhythmogenesis and fatal central apnea at birth. Nat Neurosci. 2003;6:1091–100. 66. Sosa-Pineda B. The gene Pax4 is an essential regulator of pancreatic beta-cell development. Mol Cells. 2004;18:289–94. 67. Kordowich S, Collombat P, Mansouri A, Serup P. Arx and Nkx2.2 compound deficiency redirects pancreatic alpha- and beta-cell differentiation to a somatostatin/ghrelin co-expressing cell lineage. BMC Dev Biol. 2011;11:52. 68. Collombat P, Hecksher-Sorensen J, Broccoli V, Krull J, Ponte I, Mundiger T, et al. The simultaneous loss of Arx and Pax4 genes promotes a somatostatin-producing cell fate specification at the expense of the alpha- and beta-cell lineages in the mouse endocrine pancreas. Development. 2005;132:2969–80. 69. Price M, Lazzaro D, Pohl T, Mattei MG, Ruther U, Olivo JC, et al. Regional expression of the homeobox gene
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REVIEW ARTICLE
Pancreatic regeneration: basic research and gene regulation Kenji Okita1 · Toru Mizuguchi1 · Ota Shigenori1 · Masayuki Ishii1 · Toshihiko Nishidate1 · Tomomi Ueki1 · Makoto Meguro1 · Yasutoshi Kimura1 · Naoki Tanimizu3 · Norihisa Ichinohe3 · Toshihiko Torigoe2 · Takashi Kojima4 · Toshihiro Mitaka3 · Noriyuki Sato2 · Norimasa Sawada5 · Koichi Hirata6
Received: 4 February 2015 / Accepted: 19 May 2015 © Springer Japan 2015
Abstract Pancreatic regeneration (PR) is an interesting phenomenon that could provide clues as to how the control of diabetes mellitus might be achieved. Due to the different regenerative abilities of the pancreas and liver, the molecular mechanism responsible for PR is largely unknown. In this review, we describe five representative murine models of PR and thirteen humoral mitogens that stimulate β-cell proliferation. We also describe pancreatic ontogenesis, including the molecular transcriptional differences between α-cells and β-cells. Furthermore, we review 14 murine models which carry defects in genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.
* Toru Mizuguchi [email protected] 1
Department of Surgery, Surgical Oncology, Sapporo Medical University, Sapporo, Hokkaido 060‑8543, Japan
2
Department of Pathology I, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
3
Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
4
Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
5
Department of Surgical Pathology II, Sapporo Medical University, Sapporo, Hokkaido 060‑8556, Japan
6
Department of Surgery, JR Sapporo Hospital, N‑3, E‑1, Chuo‑Ku, Sapporo, Hokkaido 060‑0033, Japan
Keywords Pancreatic regeneration · Transcriptional factors · Gene regulation
Introduction Pancreatic regeneration (PR) is an interesting phenomenon which could provide clues as to how the control of diabetes mellitus might be achieved. The pancreatic functions can be divided into exocrine and endocrine functions; however, most research into the physiology and morphology of islet cells has focused on β-cells, which only perform endocrine functions. Despite the fact that liver regeneration has been extensively investigated [1–3], little is known about PR. The liver and pancreas both develop from the endoderm and share a lot of gene regulation mechanisms; however, they have markedly different regenerative abilities. In this review, we summarize the murine models of PR and review the humoral mitogens for β-cell proliferation and pancreatic ontogenesis. We also review the murine models that carry defects in the genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.
Models of pancreatic regeneration PR is known to occur in the neonatal period in humans, as well as in type I diabetes, obesity, and pregnancy. However, a comprehensive study of human PR after 50 % resection involving 13 patients who underwent two consecutive partial pancreatic resections did not obtain any evidence of β-cell regeneration during an observation period of 1.8 ± 1.2 years [4]. Thus, a high incidence of diabetes is probably inevitable after pancreatectomy. Although human
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PR could be totally different from the PR seen in other species, numerous animal models have been developed to investigate the molecular and physiological mechanisms of PR. Scow et al. tried to produce a rat model of total pancreatectomy to investigate the physiological mechanism responsible for the acute onset of diabetes. However, they found that it was difficult to perform total pancreatectomy in rats. In addition, the rats did not develop diabetes and remained alive and in good health for several months [5]. Anatomical peculiarities are the main reason why it is technically difficult to perform total pancreatectomy in rats. Although only a very small amount of tissue is left in the area between the duodenal wall and bile duct after total pancreatectomy, it is large enough to enable the rats to maintain glucose homeostasis. Due to these difficulties, Houry et al. reported a modified total pancreatectomy technique for rats which involved a liver transplant [6], and Migliorini et al. described a two-stage rat total pancreatectomy procedure [7]. Wenger et al. attempted to develop a rat model of surgically induced diabetes by making further modifications to the above surgical procedures because previous models exhibited incomplete diabetes after surgery [8]. Partial pancreatectomy has also been used to investigate β-cell physiology. The anatomical nomenclature for rat partial pancreatectomy was first described in 1964 [9]. Subsequently, Pearson et al. observed PR in rat models subjected to 52, 69, or 91 % pancreatectomy [10]. The 52 % model was considered to represent splenic resection and was renamed the 50 % model, the 69 % model was considered to represent gastrosplenic resection and was referred to as the 70 % model, and the 91 % model was considered to represent subtotal resection and was renamed the 90 % model. Partial pancreatectomy stimulated PR in each of these models. Specifically, it caused 21, 32, and 78 % increases in the pancreatic weight in the 50, 70, and 90 % models, respectively (Table 1). Pancreatic duct ligation (PDL) is another experimental model of PR. Although PDL has been extensively studied, it has not been conclusively determined whether it promotes PR with the involvement
of both exocrine and endocrine cells [11]. PDL has been reported to induce the development of putative pancreatic precursor cells, which express neurogenin 3 (Ngn3) [12], carbonic anhydrase II [13], and pancreas-specific transcription factor 1 subunit alpha (Ptf1a) [14]. However, in another study β-cell regeneration was not observed after PDL [11].
Humoral growth factors for β‑cells Several humoral growth factors have been shown to act as mitogens to stimulate β-cell proliferation. Betacellulin was discovered by Shing et al. in 1993. It is a member of the epidermal growth factor (EGF) family and exhibits 50 % homology with transforming growth factor (TGF)-α [15]. Betacellulin was identified as a 32-kilodalton (kDa) glycoprotein in conditioned medium derived from mouse pancreatic β-cell tumors. The injection of betacellulin ameliorated glucose intolerance in an alloxan-induced mouse model of diabetes by increasing β-cell volume [16]. Other members of the EGF family, including EGF itself [17], TGF-α [18], and keratinocyte growth factor [19], also act as β-cell mitogens. In addition, hepatocyte growth factor (HGF) has been shown to have strong post-injury mitogenic effects in multiple organs. Although it is uncertain whether the administration of HGF promotes β-cell proliferation, HGF has been shown to promote β-cell proliferation after partial pancreatectomy and streptozocin-based β-cell ablation [20]. Glucagon-like peptide-1 (GLP-1) and exendin-4, which are incretins, are also considered to β-cell mitogens [21]. These proteins have similar structures and bind to the GLP-1 receptor [22]. The glucose-dependent insulinotropic polypeptide is another incretin that promotes β-cell proliferation and controls glucose homeostasis [23]. Throughout the world, long-acting incretin analogs are now widely used in combination with inhibitors of the degrading enzyme dipeptidyl peptidase-4 to treat type II diabetes [24]. Gastrin can also stimulate β-cell neogenesis and the proliferation
Table 1 Murine pancreatectomy models Operation
Author
Years
Journal
Techniques
Result
Total pancreatectomy
Scow
1956
Endocrinology
Total pancreatectomy
No diabetes
Houry
1983
Eur Surg Res
Transplant technique Total splenopancreatectomy
Diabetes after 8 h
Migliorini
1970
Diabetes
Two-stage technique
Diabetes after 24 h
Wenger
1990
J Surg Res
Radical splenopancreatectomy
Diabetes
Pearson
1977
Gastroenterology
52 % (rounded off to 50 %): splenic resection
21 % increase in pancreatic weight
69 % (rounded off to 70 %): gastrosplenic resection
32 % increase in pancreatic weight
91 % (rounded off to 90 %): subtotal resection
78 % increase in pancreatic weight
Partial pancreatectomy
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Description
Betacellulin Epidermal growth factor Transforming growth factor–α Keratinocyte growth factor Growth factors Hepatocyte growth factor/c-met Incretins Glucagon-like peptide-1 Exendin-4 Glucose-dependent insulinotropic polypeptide
suggests the existence of other proteins with similar functions to betatrophin [35] (Table 2).
EGF family
Others
Gastrin Glucagon-like peptide/gastrin Epidermal growth factor/gastrin Islet neogenesis-associated protein pentadecapeptide Mangiferin Silymarin Betatrophin
of β-cell masses from transdifferentiated pancreatic exocrine cells [25]. In addition, the combined administration of gastrin and GLP-1 or EGF resulted in greater mitogenic activity in comparison to that observed with the administration of a single mitogen [26]. Islet neogenesis-associated protein pentadecapeptide (INGAP-PP) is a section of the INGAP protein (amino acids 104–118) that was discovered in 1992 [27], and which has been experimentally used in the treatment of type I and type II diabetes in a randomized double-blinded clinical study [28]. Although it is not yet commercially available for clinical use, it was shown to modestly improve the clinical parameters of the diabetes patients to whom it was administered [28]. Mangiferin is a natural xanthonoid that can be extracted from mangos and which is commercially available [29]. As well as acting as a β-cell mitogen, mangiferin possesses multiple other functions, such as anti-oxidant, insulin-stimulating, and antiviral activities [29]. Silymarin is a herbal medicine that is derived from Silybum marianum (milk thistle) [30, 31]. Seven flavonolignans, including silybin (which accounts for 50–70 % of the flavonolignans found in S. marianum), and the flavonoid taxifolin can be extracted from S. marianum [31]. Soluble biosynthesized silybin has been developed in order to increase the bioavailability of the molecule [31]. The administration of silymarin resulted in increased serum insulin levels and β-cell neogenesis in a partially pancreatectomized rat model [32]. Betatrophin (also known as ANGPTL8) is an angiopoietin-like protein that exhibits some functional overlap with ANGPTL3 [33]. Although betatrophin promotes the proliferation of pancreatic β-cells and increases insulin secretion [34], betatrophin knockout (KO) mice are born normally and develop lean bodies. This
Pancreatic ontogenesis The pancreas develops from the foregut endoderm. Its development is initially associated with the activation of pancreatic and duodenal homeobox 1 (Pdx1) [36, 37]. In addition, sonic hedgehog (Shh) determines the fate of duodenal/pancreatic precursor cells [38]. The turning off of Shh expression and the upregulation of Ptf1a expression are necessary for the differentiation of duodenal/pancreatic precursors into pancreatic precursors; otherwise, they follow an intestinal fate [36]. The further induction of Ptf1a results in the differentiation of pancreatic precursors into exocrine cells. On the other hand, the activation of Ngn3 encourages pancreatic precursors to become endocrine progenitors [39]. Human transcription factor 2 (TCF2; also known as vHNF1 or HNF1β) is a POU-homeobox transcription factor that represses Shh and activates Ptf1a [40]. TCF2 also activates Ngn3, islet-1 (Isl1), and Pax6 [40]. The turning off of hepatocyte nuclear factor 6 (HNF-6) and Ptf1a expression is required for the differentiation of pancreatic precursors into endocrine cells [36]. On the other hand, the musculoaponeurotic fibrosarcoma oncogene family (Maf)B induces the transformation of endocrine cells into α-cells and β-cells [41]. The aristaless-related homeobox (Arx) and paired box 4 (Pax4) genes mutually regulate each other’s expression and determine the final functions of endocrine cells. The induction of Arx is seen in α-cells and ε-cells. On the other hand, upregulated Pax4 expression is observed in β-cells and δ-cells. Furthermore, it was demonstrated that brain-4 (Brn4) and Iroquoisrelated homeobox gene 2 (Irx2) are specifically expressed in α-cells, whereas the Nirenberg and Kim homeobox (Nkx) genes (Nkx 2.2, Nkx 6.1, and MafA) are specifically expressed in β-cells (Fig. 1).
Mouse KO models of key transcription factors for pancreatic ontogenesis PDX1 KO mice Pdx1 (also known as IPF1, IDX-1, and STF-1) is a homeobox-like homeoprotein that plays a crucial role in pancreatic development [42]. The homozygous depletion of Pdx1 results in the complete absence of the pancreas and death within a few days of delivery [43]. Heterozygous depletion is not lethal, but causes diabetes and a reduction in the number of β-cells [44] (Table 3).
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Fig. 1 Pancreatic ontogenesis and transcriptional gene regulation. Pdx1 pancreatic and duodenal homeobox 1, Shh sonic hedgehog, Ptf1a pancreasspecific transcription factor 1 subunit alpha, TCF transcription factor, vHNF1 variant hepatocyte nuclear factor, HNF hepatocyte nuclear factor, Ngn3 neurogenin 3, Isl1 Islet-1, Maf Musculoaponeurotic fibrosarcoma oncogene family, Pax paired box, Nkx Nirenberg and Kim homeobox, Arx Aristalessrelated homeobox gene, Brn brain, Irx Iroquois-related homeobox gene, PP-cells pancreatic polypeptide-producing cells
Endoderm Pdx1 on Duodenal/Pancreatic precursors Duodenal mucosa
Shh on
Shh off Ptf1a on
TCF2 (vHNF1, HNF1β)
Pancreatic precursors Endocrine progenitors
Ptf1a on high
Exocrine cells
Endocrine cells
Ngn3 on Isl1 on HNF6 off Ptf1a off
MafB Arx on Pax6 on
α-cells glucagon
ε−cells ghrelin
Brn4 Irx2
Pax4 on Nkx2.2, 6.1 Pdx1 on high β-cells insulin
δ-cells somatostatin
γ-cells (PP-cells) pancreatic polypeptide
MafA
Table 3 Mouse knockout models of key transcription factors for pancreatic organogenesis Genetic defect
Morphology
Homozygous phenotype
Pdx1 Ptf1a (PTF1-p48) Sonic hedgehog (Shh)
Reduced insulin and glucagon production Pancreatic agenesis Severe lung branching defects Essential for coronary vascular development
No pancreas, death occurs within a few days Death occurs shortly after birth Death occurs during the embryo stage
TCF2/vHNF1
Defective visceral endoderm formation Pancreatic agenesis by embryonic day 13.5
Death occurs during the embryo stage
Ngn3
Normal at birth, but death occurs after 2–3 days due to dehydration
Death occurs 2–3 days after birth
Isl1 HNF6
The dorsal pancreatic mesenchyme does not form Reduction in the number of endocrine cells
MafB KO Pax4
Reduced numbers of α- and β-cells Increased production of glucagon and ghrelin Decreased numbers of β- and δ-cells
Death occurs during the embryo stage Growth retarded and 75 % die between 1 and 10 days after birth due to liver failure Death occurs at birth due to central sleep apnea Normal appearance, but die within 3 days
Arx
Increased numbers of β- and δ-cells No mature α-cells
Death occurs within 2 days after birth
Nkx2.2
No β-cells, but precursor cells survive Few α- or PP-cells
Death occurs shortly after birth
Nkx6.1 Pax6
No β-cells (including precursors) No α-cells, but other cell types are present
Death occurs immediately after birth Death occurs a few minutes after birth
MafA
Normal pancreas, but age-dependent functional defects
MafA deficiency does not confer embryonic lethality
Pdx1 pancreatic and duodenal homeobox 1, Ptf1a pancreas-specific transcription factor 1 subunit alpha, TCF transcription factor, vHNF variant hepatocyte nuclear factor, Ngn3 Neurogenin 3, Isl1 Islet-1, HNF hepatocyte nuclear factor, Maf musculoaponeurotic fibrosarcoma oncogene family, Pax paired box, Arx Aristaless-related homeobox gene, Nkx Nirenberg and Kim homeobox, PP-cells pancreatic polypeptide-producing cells
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Ptf1a (PTF1‑p48) Ptf1a (also known as PTF1-P48) is a 48-kDa class B basic helix–loop–helix (bHLH) protein that acts as the DNAbinding subunit of the trimeric transcription factor PTF1 [45, 46]. Ptf1a is a key regulator of the fate of pancreatic precursors (i.e., whether they differentiate into exocrine or endocrine cells) [46]. The null mutation of Ptf1a results in death shortly after birth [47]. The exocrine pancreas was absent from embryos carrying a null mutation in Ptf1a, although all four endocrine cell lineages were present [47]. However, endocrine cells were found in the spleen and functioned until postnatal death [47].
[61], as the dorsal pancreatic mesenchyme does not form properly [62]. HNF‑6 KO mice HNF-6 is a cut-like homeodomain protein and a key component of the pancreatic transcription factor cascade [63]. The main phenotypic features of HNF-6 KO mice are as follows: (I) the development of the endocrine pancreas is severely inhibited; (II) the appearance of the islets of Langerhans is delayed; and (III) when the islets form, they display a perturbed architecture and contain cells that have not reached full maturity [64].
Sonic hedgehog (Shh)
MafB KO mice
Shh is a mammalian hedgehog gene, which is essential for the development of numerous organs [48], and which has been reported to cause the formation of an annular pancreas [49]. Shh-deficiency is lethal and results in death shortly after birth [49, 50]. Shh is expressed in the primitive gut, including the presumptive pancreatic area [49], and is repressed by activin B. Shh inhibition is required to initiate the pancreatic differentiation program [51].
MafB is also a member of the basic-leucine zipper-containing transcription factor family and is distinguished from other large Maf molecules by the presence of an N-terminal transactivation domain [41, 65]. MafB-deficient mice die at birth due to central sleep apnea [65]. MafB is a regulatory transcription factor that plays a key role in the development of mature α- and β-cells [41]. Pax4 KO mice
TCF2/vHNF1 The human transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1β (a human POU-homeobox protein also known as vHNF) [40]. A mutation in the TCF2 gene is responsible for maturity-onset diabetes of the young type 5 (MODY5) [40, 52]. TCF2 plays a crucial role in controlling pancreatic development, and Ptf1a expression is not induced in the absence of TCF2 expression [40]. In addition, null mutation of the TCF2 gene causes pancreas agenesis and death before gastrulation [40]. Ngn3 KO mice Ngn3 is a member of a family of bHLH transcription factors that are expressed in the embryonic pancreas [53]. Ngn3 mutant mice are morphologically indistinguishable from wild-type mice and feed normally after birth, but die after 2–3 days [39]. This phenotype is reminiscent of the phenotype of diabetic mice with mutations in neurogenic differentiation (NeuroD)/BETA2 [54], Pdx1 [55], Nkx 2.2 [56, 57], or Pax4 [58, 59].
Pax4 is a unique member of the Pax family of transcription factors that is exclusively expressed during the embryonic stage [66]. Although heterozygous Pax4-deficient mice have normal lifespans and are fertile, homozygous mice die within 3 days due to dehydration [59]. Arx KO mice Defects in the Arx gene result in the opposite phenotype to that seen in Pax4-deficient animals [67]; i.e., a reduced number of α-cells, and greater numbers of β- and δ-cells. Direct mutually antagonistic transcriptional repression has been shown to occur between Arx and Pax4 [68]. Nkx2.2 KO mice Nkx2.2 is a member of the vertebrate homeodomain transcription factor gene family and is almost homologous to the Drosophila NK2/ventral nervous system defective (vnd) gene [69]. Homozygous Nkx2.2 deficiency results in severe hyperglycemia and death shortly after birth [57]. Nkx2.2 is required for the final differentiation of pancreatic β-cells [57].
Islet‑1 KO mice Nkx6.1 and Nkx6.2 KO mice Isl1 is a transcriptional regulator of the LIM-homeodomain and plays a key role in the regulation of islet development [60]. Isl1 deficiency results in death during embryogenesis
Although the pancreas develops normally in Nkx6.2 singlemutant mice, Nkx6.1 mutants exhibit severe defects in their
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β-cells, including in their β-cell precursor cells [70]. In addition, Nkx6.1/Nkx6.2 double-mutant embryos display a markedly reduced number of α-cells [71]. Pax6 KO mice Pax6 is the gene responsible for human aniridia and mutant mice with small eyes [72]. Homozygous Pax6-deficient mice are born without eyes and do not develop distinct islets. In addition, they exhibit a marked deficiency of α-cells [73]. Musculoaponeurotic fibrosarcoma oncogene family A KO mice The musculoaponeurotic fibrosarcoma oncogene family (Maf)A is a basic-leucine zipper transcription factor that plays an important role in maintaining β-cell function [74]. In MafA KO mice, although the pancreas develops fully, age-dependent defects are observed in glucose homeostasis [74, 75].
Conclusion We reviewed PR, including its ontology and molecular biology, from a basic point of view. Knowledge on PR is accumulating rapidly, and the molecular regulatory mechanisms responsible for PR are being revealed. Various transcription factors are critical for pancreatic development and survival. In the future, we hope to create pancreatic cells for therapeutic purposes. Acknowledgments Part of this study was supported by a Grant-inAid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No.24659592) to T. Mizuguchi, T. Torigoe, N. Sato, and K. Hirata. Part of this study was also supported by a Health Labour Sciences Research Grant from the Ministry of Health, Labour, and Welfare (No. 2601023) to T. Mizuguchi, T. Torigoe, K. Hirata, and N. Sato.
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