1: INTRODUCTION
1.7. The pancreas, anatomy, physiology, development and functions
1.7.1. Anatomy and basic physiology
The pancreas is a solid organ that lies transversely across the posterior abdominal wall, in the retroperitoneum, in front of the abdominal aorta and first and second lumbar vertebrae. It is a gland with a double function: 1) an exocrine function to produce numbers of enzymes for digestion, which are usually in an inactive form within the pancreas and get activated in the duodenum; 2) an endocrine function to produce several hormones important in glucose homeostasis, nutrient absorption within tissues, storage and metabolism.
1.7.1.1. The exocrine pancreas
The exocrine pancreas represents 99% of the mass of the organ and is mainly composed of clusters of acinar cells secreting pro-enzymes. These enzymes are collected via ductules, into intercalated ducts and then into the main pancreatic duct of Wirsung or the accessory pancreatic duct of Santorini and reach the duodenum.
The exocrine fraction of the pancreas produces about 1.5l of pancreatic juice per day. It contains water, ions and a variety of proteins. It is alkaline due to HCO3
and serves at neutralizing the gastric acid entering the duodenum with ingested food.
Most of the proteins in pancreatic juice are enzymes and proenzymes. Some enzymes (lipase, amylase, deoxyribonuclease and ribonuclease) are secreted in their active form, whereas other enzymes (trypsinogen, chymotrypsinogen, proelastase, procarboxypeptidase and phospholipase A2) are secreted as proenzymes and are activated in the lumen of the proximal intestine. Pancreatic secretions therefore allow the digestion of lipids, carbohydrates and proteins within the intestine.
1.7.1.2. The endocrine pancreas
The endocrine pancreas is composed of multiple structures forming the islets of Langerhans, which are distributed throughout the exocrine pancreas. Islets of Langerhans represent approximately 1% of the total mass of the pancreas (Kahn, 2008). There are about 1 million islets in a human pancreas and each islet contains several hundreds of cells (Cabrera, 2006; Stefan, 1982). Four endocrine cell types constitute the functional entities of the islets of Langerhans: alpha cells secreting glucagon, beta cells secreting insulin, delta cells secreting somatostatin, and F or PP
cells secreting pancreatic polypeptide. The islets contain about 80% of beta cells and their architecture was analyzed extensively (Brissova, 2005; Cabrera, 2006;
Sakuraba, 2002). In rodents, beta cells are arranged within the central part of the islet surrounded by a monolayer of alpha cells (Cabrera, 2006; Gomez Dumm, 1995;
Starich, 1991). This architecture was believed to be similar in human islets but recent studies showed a random distribution of cells within a given space in larger human islets, and a “mantle-core” structure within lobular organization in smaller islets (Bonner-Weir, 2008b; Cabrera, 2006). The islets are highly vascularized structures (Ballian, 2007; Nyman, 2008). Endocrine cells sense glucose levels in the blood and respond by adequate secretions of hormones (Goren, 2005). The venous drainage flows into the portal vein and thereby islet-secretory products pass directly into the liver, a major site of action of glucagon and insulin (gluconeogenesis, glycogenolysis, etc). The islets are also innervated, by both parasympathetic and sympathetic axons.
Neural control of islet cell hormone secretion, directly through the sympathetic fibers and indirectly through stimulation by catecholamine released from the adrenal medulla plays an important role in glucose homeostasis during stress (Dunning, 1991).
1.7.1.3. The beta cell
The beta cell synthesizes and secretes insulin in order to regulate glucose homeostasis in the body. When this function is deficient, or when islets are destroyed, the patient suffers from a disease called diabetes mellitus. Insulin is a protein composed of two peptide chains (A and B chains), that are connected by two disulfide bonds. Insulin is synthesized in beta cells first as a precursor, preproinsulin which is cleaved into proinsulin. Within secretory granules, proinsulin is cleaved at two sites to form insulin (51 amino acids) and the biologically inactive C peptide. Both are secreted into blood stream following beta cell stimulation.
Briefly, when glucose concentrations increase within blood, glucose enters beta cells through low affinity glucose transporters (GLUT)-2 (Thorens, 2001). After its entry, glucose is phosphorylated into glucose-6-phosphate by glucokinase, the true glucose sensor (Bedoya, 1986; Matschinsky, 1996). ATP produced during glucose metabolism causes the closure of ATP sensitive potassium channels (KATP), leaving the background sodium entry without balance. This depolarizes the cell and allows calcium entry, which triggers insulin release (illustrated in figure 4) (Ashcroft, 1994;
Newgard, 1995).
Glucose is not the only stimulator for insulin release. Other factors such as amino acids (i.e. arginine) (Jarrousse, 1975), vagal stimulation (Zawalich, 1989), and enteric hormones (i.e. GLP-1) (MacDonald, 2002) also induce insulin secretion. Insulin itself inhibits its secretion (Elahi, 1982).
Insulin will then act on multiple organs through the insulin receptor. Insulin is an anabolic hormone and acts on: 1) the liver: increase in glycogenesis and lipid synthesis, reduction in glycogenolysis, gluconeogenesis, fatty acid oxidation; 2) muscle: increase in glucose uptake and glycogenesis, reduction in protein catabolism; 3) adipose tissue: increase in lipoprotein lipolysis and fatty acid esterification, reduction in lipolysis of stored fat.
The beta cell within the islet interacts closely with other beta cells but also with other cells composing islets and the extracellular matrix. These interactions influence insulin secretion and survival of beta cells in vitro (Bosco, 2000; Bosco, 1989;
Hammar, 2004; Hammar, 2005; Parnaud, 2006).
Figure 4: beta cell function in response to glucose
Stimulus-secretion coupling in beta cells. Glucose enters through low affinity glucose transporters (GLUT2). Glucose metabolism increases ATP within the cells closing potassium channels and leading to cell depolarization. This results in opening of the voltage dependent Ca2+channels. Ca2+ induces secretion of insulin through fusion of granules containing insulin with the plasma membrane. ER:
endoplasmic reticulum, KATP: ATP sensitive potassium channel, TRP: transient receptor potential channel, Kv: voltage gated potassium channel. Adapted from Hiriart, M et al. (Hiriart, 2008).
Glucose
GLUT2 KATP channels
TRPs
T-type Ca2+
channels
Insulin receptor
Kv Channels G-coupled
receptor Receptor for
neurotransmitter
Ca2+
Na+ channels
L-type Ca2+
channels
1.7.2. Pancreas development
The pancreas is derived from the endoderm. In rodents at E 8.5 and in humans on day 26, a duodenal bud starts to grow into the dorsal mesentery, and will form the dorsal pancreatic bud. In humans, few days later, as the dorsal pancreatic bud elongates into the dorsal mesentery, another endodermal diverticulum grows into the ventral mesentery, just distal to the developing gallbladder, the ventral pancreatic bud (Gittes, 2009; Slack, 1995). This bud fuses with the proximal end of the common bile duct on day 32 in humans. Both ventral and dorsal pancreatic buds thicken and form multilayered solid buds within the duodenal portion of the developing intestine (Gittes, 2009; Slack, 1995). These epithelial clusters then form intraepithelial microlumens which coalesce and generate continuous lumens, thereby forming and epithelial tree, with acinar cells and ductal cells. This tree forms the drainage system, the pancreatic duct, which transports digestive enzymes from acinar cells to the duodenum. Islets also form from these epithelial buds and upon differentiation delaminate and aggregate into clusters in which they continue to proliferate until birth and early childhood in human beings (Kassem, 2000).
Similar to liver development, interactions between pancreatic endoderm with surrounding mesoderm plays an important role in specifying exocrine and endocrine pancreatic lineages. This adjacent mesoderm secretes soluble factors like FGFs, BMPs, activin A, and retinoic acid (Dessimoz, 2006; Kumar, 2003; Stafford, 2004).
Within pancreatic precursors, sequential expression of transcription factors in response to these stimuli induces differentiation into the various types of endocrine cells. The pancreatic epithelium while expanding and branching into the dorsal and ventral mesenteries, expresses uniformly pancreatic and duodenal homeobox 1 gene (Pdx1) and pre-B-cell leukemia homeobox 1 (Pbx1). This expression subsequently induces expression of neurogenin3 (Ngn3) specifically in endocrine cells, (Schwitzgebel, 2000). Transcription factor expression is precisely regulated on the time axis during differentiation into endocrine cells (Soria, 2001) and (Habener, 2005). Final differentiation is induced secondary to expression of specific transcription factors and characterized by expression of specific proteins such as insulin, glucokinase, Glut-2 in beta cells, and other hormones such as glucagon in alpha cells, somatostatin in delta cells and PP in PP cells. During beta cell differentiation, the following transcription factors are expressed: Pdx1, Isl1, Ngn3, NeuroD, Pax6, Pax4, Nkx2.2, Nkx6.1, MafA (see figure 5 A and B). Expression of
early and late factors was analyzed to identify potential beta cell progenitors or to evaluate the induction of beta cell differentiation in stem cells (D'Amour, 2006).
Figure 5: expression of transcription factors during endocrine development in mice A
B
A) Sequential expression of transcription factors during beta cell development, adapted from Soria, B.
(Soria, 2001). B) A schematic representation of transcription factor expression during development based on the temporal expression and phenotypic results of gene-specific knockouts of the factors in mice. Adapted from Habener et al. (Habener, 2005)
1.7.3. Diabetes
Diabetes mellitus is more a description of a symptom than a disease. It characterizes the clinical presentation of hyperglycemia (diagnosed by a fasting glucose
>7.0mmol/l on tow occasions or a glucose level >11.1mmol/l, 2 hours after ingestion of 75g of glucose) (Mayfield, 1998). Hyperglycemia in all cases is due to a functional deficiency of insulin action. Deficient insulin action can be due to a decrease of insulin secretion, a decreased response to insulin by target tissues (insulin resistance) or an increase in the counter-regulatory hormones. Diabetes can be divided into type 1, or insulino-dependent diabetes, and type 2, non-insulino-dependent diabetes. Type 2 diabetes, the most common type of diabetes, is characterized by insulin resistance. It is associated with metabolic syndrome in Western countries and has a much stronger genetic component (Doria, 2008).
Type 1 diabetes is characterized by the destruction of beta cells in the pancreas. In absence of insulin, hyperglycemia results in polyuria, polydipsia and weight loss.
Ketone bodies are also increased due to the lack of insulin, resulting in severe life-threatening acidosis. Physiopathology of type 1 diabetes is not fully understood yet, but it is associated with genetic predispositions, related to MHC class2 (HLA-DQ) (Onengut-Gumuscu, 2002), auto-immune disease (Bach, 1994; Eisenbarth, 1986), and environmental factors which induce a stress for the body, such as viral infections (Atkinson, 2001; Jun, 2003). These multifactorial etiologies cause inflammation of the islets and infiltration of various cell types such as macrophages and T-cells. During inflammation, cytokines are released within the islets (TNF-alpha, Il1-beta, NO, Ifn-gamma, etc.), cytotoxic T-lymphocytes attack islets cells and B-lymphocytes produce auto-antibodies specific for beta cells components (Cnop, 2005; Franke, 2005;
Kaminitz, 2007). Beta cells thereby die either from apoptosis, from T-cell attack or from antibody-mediated cytotoxicity.
Presently, no treatment is available to prevent islet destruction or reverse the disease once it has become symptomatic. Immunologic approaches are under active investigations to prevent beta cells destruction by auto-reactive T-cells and some clinical studies are actively recruiting patients (Belghith, 2003; Chatenoud, 2007;
Herold, 2005; Mallone, 2007; Martinuzzi, 2008).
Since the introduction of subcutaneous exogenous injections of insulin, survival of patients suffering from diabetes has been improved and prolonged. Nevertheless, insulin-therapy does not prevent long term complications, secondary to macro- and microangiopathy (1993; 2000; Fox, 2004; Nathan, 1993). Intensive glycemic control
can improve long term survival and decrease complications, but the patients are at increased risk of hypoglycemia (1993; 2000).
Currently, pancreas or islet transplantation is an alternative treatment to insulin injections (Stock, 2004). Despite the success of islet transplantation (achieved with the Edmonton protocol) (Shapiro, 2000) an in pancreas transplantation (Gruessner, 2005), the chronic lack of organ donors and the world wide number of patients suffering from type 1 diabetes motivates the search for alternative therapies, such as stem cells for cell therapy (Efrat, 2008; Hogan, 2008).
1.7.4. Pancreatic and beta cell regeneration/proliferation
In adult humans, little evidence exists that beta cells can regenerate (Jun, 2008).
However, in children suffering from nesidioblastosis, a disease characterized by diffuse and disseminated proliferation of primitive beta cells within the pancreas resulting in hyperinsulinemia and profound hypoglycemia, subtotal pancreatectomy is often the treatment of choice. A follow-up study showed that in more than 50% of children, pancreatic tissue regenerated and no diabetic symptoms were observed (Berrocal, 2005). In rodents, pancreas regeneration has been documented (Brennand, 2007; Dor, 2004). Multiple studies showed that pancreas regeneration occurs after surgical subtotal pancreatectomy (De Leon, 2003; Xu, 1999). Recently, using a transgenic mouse model, in which most beta cells were destroyed by beta cell specific inducible expression of diphtheria toxin A gene and administration of diphtheria toxin B, beta cell regeneration occurred after toxin withdrawal, with normalization of glycemia (Nir, 2007). Cell proliferation of surviving beta cells was identified as the major mechanism. Similar results were obtained in another transgenic mouse model in which a tamoxifen inducible c-Myc transcription factor /mutant estrogen receptor (c-MycERTAM) was used to induce apoptosis in beta cells (Cano, 2008).
Multiple theories have been described to explain the origin of beta cells in the regenerating pancreas. Four major sources of new beta cells have been suggested:
1) replication of preexisting beta cells, 2) differentiation of stem/progenitors in the ductal epithelium 3) acinar transdifferentiation to beta cells 4) differentiation of stem/progenitors (different from acinar, ductal or islet cells) (Bonner-Weir, 2005).
Figure 6: pancreas as a source of new beta cells
Four different theories regarding the origin of beta cells during pancreas regeneration. Adapted from Bonner-Weir S et al (Bonner-Weir, 2005).
As mentioned before, recent lineage tracing studies performed in mice demonstrated that most beta cells are generated through replication from preexisting beta cells (Cano, 2008; Dor, 2004; Nir, 2007). Beta cell proliferation is increased by the addition of growth factors such as Exendin-4 or GLP-1 (De Leon, 2003; Xu, 1999) and replication is cyclin-D2 dependent, as mice deficient in cyclin-D2 showed hyperglycemia and reduced beta cell mass (Georgia, 2004). In ex vivo culture experiments, rodent islets and beta cells proliferated as shown by BrdU incorporation and Ki67 expression (Parnaud, 2008).
Other studies, however, showed endocrine marker expression within ductal structures and differentiation into beta cells, after pancreatic injury or selective destruction of islets (Bonner-Weir, 2008a; Bonner-Weir, 2004; Rooman, 2002).
A recent study showed that when acinar cells were infected in vivo with Ngn3, Pdx1 and Mafa, three beta cell transcription factors, using an adenovirus vector, they showed expression of beta cell markers, insulin secretion and reversal of hyperglycemia. This technology of cellular reprogramming using defined factors demonstrates that acinar cells, when reprogrammed, can become functional endocrine cells and that gene therapy could be used for the induction of transdifferentiation and cellular replacement (Zhou, 2008b).