**4. Role of the exocrine pancreas in digestion**

Pancreas plays a crucial role in digestion. Its exocrine part secretes enzymes that are involved in digestion of carbohydrates, proteins and lipids. In this section, we will briefly review the digestive processes in general, and specifically point out the contribution of the pancreatic juice.

#### **4.1. Assimilation of carbohydrates**

In the western diet, the mean daily intake of carbohydrates is about 300 g that yield about 1200 kcal in metabolism [124]. Starch, the plant storage polysaccharide, constitutes by far the largest percentage of the carbohydrate intake (70%). About 30% of starch is composed of amylose (a straight polymer of glucose), the remainder of amylopectin (a branched polymer of glucose) [125, 126]. These different constituents of starch require different enzymes for their cleavage. Further 20% of carbohydrates in the food contribute refined sugars (e.g. sucrose, fructose and glucose) and approximately 10% the disaccharide lactose from various sources [127].

Digestion of carbohydrates takes place in two subsequent steps that are separated spatially: (i) digestion in the lumen and (ii) at the enterocyte brush border. Acinar cells of salivary glands (parotid, sublingual and submandibular) and of exocrine pancreas all produce and secrete the closely related enzyme *α*-amylase [128, 129]. *α*-amylase is secreted in an active form and has an optimal pH for enzymatic activity at pH = 7.0 [130]. It is an endo-enzyme that cleaves internal *α*-1,4 glycosidic bonds, but not *α*-1,6 bonds, terminal *α*-1,4 bonds, or α-1,4 bonds that are next to the branches in the molecular structure [131]. Amylopectin is thus cleaved to maltose and *α*-limit dextrins, whereas amylose is cleaved to maltose and maltotriose. The reactions seem to yield also a small percentage of free glucose [131].

The meal response of pancreatic secretion can be divided into four phases, i.e. cephalic, gastric, intestinal and inter-digestive phases. The names correspond to the origin of the predominant form of pancreatic secretion control. There is however significant overlap and integration between these regulatory mechanisms in response to a meal. Average pancreatic secretion stimulated by a meal amounts to about 50–60% of the maximum output of the gland [120–122].

The cephalic phase contributes approximately 25% of the meal-response secretion. It is caused by input of visual, olfactory, gustatory and tactile (mastication) sensory modalities. Sham feeding (a process, in which people are allowed to see, smell, taste but not swallow the food) causes pancreatic secretion that is rich in enzymes but low in concentration and volume of

The gastric phase accounts for about 10% of the meal-response secretion and starts with arrival of food into the stomach. It is regulated by enteropancreatic, vagovagal reflexes, which are

The majority of the meal-response pancreatic secretion occurs in the intestinal phase. The secretory output amounts to 50–60% of maximal capacity [120]. It starts as the acidic chyme from the stomach, passes into the intestine, where components of the chyme, HCl, bile and bile salts elicit hormonal and neural responses. Hormonal influence on pancreatic secretion in the intestinal phase is mainly the result of CCK and secretin and the amplification of the secretory response by neural influences of the enteropancreatic reflex [121]. Passing of stomach contents into the intestine lowers the luminal pH, which is a signal for the duodenal S cells to

<sup>−</sup> secretion from the

release secretin. It, in turn, functions as the main secretagogue for HCO3

**4. Role of the exocrine pancreas in digestion**

ductal cells and also as an enhancer of enzyme secretion from the acinar cells [77, 102].

Pancreas plays a crucial role in digestion. Its exocrine part secretes enzymes that are involved in digestion of carbohydrates, proteins and lipids. In this section, we will briefly review the digestive processes in general, and specifically point out the contribution of the pancreatic

In the western diet, the mean daily intake of carbohydrates is about 300 g that yield about 1200 kcal in metabolism [124]. Starch, the plant storage polysaccharide, constitutes by far the largest percentage of the carbohydrate intake (70%). About 30% of starch is composed of amylose (a straight polymer of glucose), the remainder of amylopectin (a branched polymer of glucose) [125, 126]. These different constituents of starch require different enzymes for their cleavage. Further 20% of carbohydrates in the food contribute refined sugars (e.g. sucrose, fructose and

glucose) and approximately 10% the disaccharide lactose from various sources [127].

<sup>−</sup> [88]. It amounts to a response that is up to 50% of maximum secretory capacity. The main mediator of the cephalic phase is the vagus nerve with ACh as the dominant neuro-

HCO3

juice.

transmitter [123].

32 Challenges in Pancreatic Pathology

stimulated by gastric distension [121].

**4.1. Assimilation of carbohydrates**

**Figure 4.** Assimilation of carbohydrates. (A) A schematic representation of the gastrointestinal tract. The origin of digestive enzymes is depicted in yellow and the location of carbohydrate absorption in beige. (B) The luminal phase of digestion critically depends on α-amylase. (C) The brush border phase involves enzymes embedded in the apical membrane of enterocytes. Monosaccharides are then transported across the epithelium by transport proteins in the apical and the basolateral enterocyte membrane.

The digestion of carbohydrates starts with intra-luminal digestion in the oral cavity. This appears not to have an important physiological role, since the salivary *α*-amylases are mostly inactivated in the acid milieu of the gastric lumen [130]. The activity of salivary *α*-amylases is partly rescued by occupying the active site of the enzyme with the substrate [132]. The digestion continues in the duodenum with the activity of pancreatic *α*-amylase, which has a rather neutral pH optimum, brought about by alkalinization of acidic chyme from the stomach by duodenal bicarbonate secretion. Contribution of the salivary amylase in starch degradation remains controversial and may become quantitatively more important when mastication time is prolonged [129, 133].

Following the intra-luminal digestion, membrane-bound enzymes of the enterocyte brush border degrade oligosaccharides produced by the luminal digestion to monosaccharides that are then absorbed into enterocytes [134]. The apical membrane of enterocyte brush border contains four major enzymes that act on the luminal side: (i) lactase, (ii) glucoamylase (maltase), (iii) isomaltase and (iv) sucrase [135]. The latter two are located on the same polypeptide chain with two distinctive active sites and are often referred to as the sucrase-isomaltase complex. An essential enzyme for starch digestion is the isomaltase, which is the only enzyme capable of degrading the α-1,6 bond, whereas the other (sucrase, glucoamylase and lactase) are involved in degrading internal α-1,4 bonds. The disaccharides sucrose and lactose are digested by the sucrase and the lactase. The final products of luminal and brush border digestion are the monosaccharides glucose, galactose and fructose, which are then absorbed through the apical membrane via SGLT-1 (glucose and galactose) and GLUT5 (fructose) transporters and through the basolateral membrane via GLUT2 [136–138]. Additionally, GLUT5 transporter in the basolateral membrane may serve as an alternative exit route for fructose (**Figure 4**).

#### **4.2. Assimilation of proteins**

Intake of proteins in the western diet amounts to approximately 70–100 g/day, which accounts for 300–400 kcal [124]. In contrast to the carbohydrates, protein digestion starts in the stomach, as virtually no significant proteolytic enzymes are found in saliva. The chief (zymogenic, peptic) cells of the gastric glands synthesize and secrete the pro-enzyme pepsinogen, the inactive precursor of pepsin, which is a proteolytic enzyme specifically suited to act in the acidic gastric milieu [139]. At pH < 5 in the gastric lumen, pepsinogen is spontaneously converted into the active form, pepsin, by cleavage of an N-terminal peptide. At pH values >5.0 and >7.5 pepsin inactivates reversibly and irreversibly, respectively [140]. Pepsin has its pH optimum at pH = 1.5–2.5. Pepsin functions as an endopeptidase, yielding oligopeptides and amino acids [141, 142].

Bulk proteolysis occurs in the small intestine. The pancreatic acinar cells secrete into the duodenum five major proteolytic proenzymes: trypsin, chymotrypsin, elastase and carboxypeptidase A and B [143–145]. First, the proenzyme trypsinogen is activated by the membranebound enterokinase to its active form trypsin [146]. The specific expression of enterokinase serves to limit proteolysis to the lumen of the small intestine. Trypsin, in turn, activates additional trypsin molecules and also converts the other four proenzymes, i.e. chymotrypsinogen, proelastase, as well as procarboxypeptidase A and B to their active forms. Carboxypeptidase A and B are ectopeptidases and cleave amino acids at the C-terminus, whereas trypsin, chymotrypsin, and elastase are endopeptidases that cleave polypeptides at specific sites resulting in 2–6 amino acid oligopeptides [144]. The oligopeptides from the lumen are further cleaved by both brush border-bound and intracellular cytosolic peptidases [134].

**Figure 5.** Protein assimilation. (A) A schematic representation of the gastrointestinal tract. Red denotes the start of digestion in the stomach, whereas beige indicates the site of digestion by pancreatic enzymes (indicated in yellow) as well as absorption. (B) Gastric luminal digestion of proteins by pepsin. (C) Intestinal luminal digestion of proteins by trypsin, chymotrypsin, elastase and carboxypeptidase A and B. (D) Brush-border assimilation of proteins involves membrane-bound peptidases, as well as peptone and amino acid (AA) transport mechanisms.

The absorption of oligopeptides and free amino acids differs. The oligopeptides are absorbed through the apical membrane with the PepT1, a H+ /oligopeptide co-transporter driven by an H+ gradient generated by the NHE3 (Na/H exchanger type 3) [147, 148]. The amino acid absorption at the apical and basolateral membrane involves at least seven and five different transport systems, respectively; however, a detailed description of these systems is beyond the scope of this chapter [149, 150] (**Figure 5**).

#### **4.3. Assimilation of lipids**

The digestion of carbohydrates starts with intra-luminal digestion in the oral cavity. This appears not to have an important physiological role, since the salivary *α*-amylases are mostly inactivated in the acid milieu of the gastric lumen [130]. The activity of salivary *α*-amylases is partly rescued by occupying the active site of the enzyme with the substrate [132]. The digestion continues in the duodenum with the activity of pancreatic *α*-amylase, which has a rather neutral pH optimum, brought about by alkalinization of acidic chyme from the stomach by duodenal bicarbonate secretion. Contribution of the salivary amylase in starch degradation remains controversial and may become quantitatively more important when mastication time

Following the intra-luminal digestion, membrane-bound enzymes of the enterocyte brush border degrade oligosaccharides produced by the luminal digestion to monosaccharides that are then absorbed into enterocytes [134]. The apical membrane of enterocyte brush border contains four major enzymes that act on the luminal side: (i) lactase, (ii) glucoamylase (maltase), (iii) isomaltase and (iv) sucrase [135]. The latter two are located on the same polypeptide chain with two distinctive active sites and are often referred to as the sucrase-isomaltase complex. An essential enzyme for starch digestion is the isomaltase, which is the only enzyme capable of degrading the α-1,6 bond, whereas the other (sucrase, glucoamylase and lactase) are involved in degrading internal α-1,4 bonds. The disaccharides sucrose and lactose are digested by the sucrase and the lactase. The final products of luminal and brush border digestion are the monosaccharides glucose, galactose and fructose, which are then absorbed through the apical membrane via SGLT-1 (glucose and galactose) and GLUT5 (fructose) transporters and through the basolateral membrane via GLUT2 [136–138]. Additionally, GLUT5 transporter in the basolateral membrane may serve as an alternative exit route for

Intake of proteins in the western diet amounts to approximately 70–100 g/day, which accounts for 300–400 kcal [124]. In contrast to the carbohydrates, protein digestion starts in the stomach, as virtually no significant proteolytic enzymes are found in saliva. The chief (zymogenic, peptic) cells of the gastric glands synthesize and secrete the pro-enzyme pepsinogen, the inactive precursor of pepsin, which is a proteolytic enzyme specifically suited to act in the acidic gastric milieu [139]. At pH < 5 in the gastric lumen, pepsinogen is spontaneously converted into the active form, pepsin, by cleavage of an N-terminal peptide. At pH values >5.0 and >7.5 pepsin inactivates reversibly and irreversibly, respectively [140]. Pepsin has its pH optimum at pH = 1.5–2.5. Pepsin functions as an endopeptidase, yielding oligopeptides

Bulk proteolysis occurs in the small intestine. The pancreatic acinar cells secrete into the duodenum five major proteolytic proenzymes: trypsin, chymotrypsin, elastase and carboxypeptidase A and B [143–145]. First, the proenzyme trypsinogen is activated by the membranebound enterokinase to its active form trypsin [146]. The specific expression of enterokinase serves to limit proteolysis to the lumen of the small intestine. Trypsin, in turn, activates additional trypsin molecules and also converts the other four proenzymes, i.e. chymotrypsi-

is prolonged [129, 133].

34 Challenges in Pancreatic Pathology

fructose (**Figure 4**).

**4.2. Assimilation of proteins**

and amino acids [141, 142].

In the western diet, 60–100 g of lipids are ingested daily, which account for about 500–900 kcal [124]. Most of the ingested lipids are in the form of triacylglycerol (TAG, 90–95%), the rest are in the form of phospholipids (PL, 5%) and cholesterol (C, < 0,5%).

Enzymes for digestion of lipids act in the watery environment of intestinal lumen, and due to their hydrophilic character, they act on the surface of ingested lipids organized in amphiphilic droplets. In the process of emulsification, larger droplets are broken into smaller ones, thereby increasing surface to volume ratio, and, consequently, enzyme efficacy [151]. Emulsification starts with food preparation, and continues in the mouth with mastication and in the gastric and intestinal lumen with churning of the ingested food. Lipid droplets organize such that the core is composed of hydrophobic TAG, whereas amphiphilic PL, C and free fatty acids (FFA) are on the surface [152, 153]. This organization, together with some proteins and carbohydrates stabilizes the products of emulsification.

Digestion of lipids exhibits a large functional redundancy [154]. It starts in the gastric lumen catalysed by the lingual and gastric lipase, together termed pre-duodenal lipase [155, 156]. The former is synthesized by acinar cells of the salivary glands and the latter by gastric chief cells. pH optimum for pre-duodenal lipase is around pH = 4, quite appropriate for the acidic gastric milieu [155, 157]. The enzyme cleaves the first ester bond in the TAG producing diacylglycerol (DAG) and an FFA [158, 159]. It is resistant to cleavage by pepsin but not by pancreatic proteases [156], and therefore functions mostly in the gastric lumen where it digests up to 15% of ingested lipids in a healthy individual. However, they might contribute to some extent to digestion of lipids in the duodenum [151, 157, 160, 161].

Digestion of lipids continues in the intestinal lumen by three major enzymes secreted by pancreatic acinar cells: the (i) pancreatic, (ii) nonspecific (carboxylic ester) lipase and (iii) phospholipase A2. The pancreatic lipase is active in the presence of a colipase (which is in turn activated by trypsin), in an alkaline pH, in the presence of Ca2+ and bile salts [162, 163]. The pancreatic lipase cleaves the first and the third ester bond in TAGs yielding 2-monoacylglycerol (MAG) and FFAs [159, 164]. Phospholipase A2 acts on the second ester bond in glycerophospholipids, giving rise to lysophospholipid (LPL) and FFAs [162]. It is secreted in an inactive form and requires for its activity an alkaline pH and bile salts. The specificity of the nonspecific lipase is low, notably, and it is able to cleave cholesterol esters and MAG [162]. The quantitative contribution of the nonspecific lipase is relatively low compared with the pancreatic lipase. During infancy, the pancreatic lipase related protein 2 contributes to digestion of lipids [165].

The luminal enzyme activity outlined above, in conjunction with an ever-ongoing emulsification, results in a multi-lamellar envelope developing around the droplet, and consisting of FFAs, bile acids, C, MAG and LPLs [166–168]. The multi-lamellar envelope bursts from the droplets in the form of vesicles that are eventually transformed to mixed micelles (especially under the influence of bile salts) that then finally serve as the main vehicle for absorption of lipids. FFAs enter the enterocyte by (i) collision with the plasmalemma and crossing of the plasmalemma by the flip-flop mechanism, or (ii) by diffusion of non-ionic fatty acids or (iii) by carrier-mediated transport. The latter probably involves the fatty acid binding protein (FABP), the fatty acid transporter protein type 4 (FATP4) and CD36 [169, 170]. MAG, LPLs and C probably enter the enterocyte by means of transport proteins or by simple diffusion. Finally, the FFAs, MAG, LPLs and C are re-esterified within the enterocyte, together with apolipoproteins assembled into chylomicrons, exocytosed into the extracellular space on the basolateral side of the plasma membrane, and reach the systemic circulation via the lymphatic circulation. In contrast, monosaccharides and amino acids reach the systemic circulation via the portal vein [151, 171] (**Figure 6**).

Enzymes for digestion of lipids act in the watery environment of intestinal lumen, and due to their hydrophilic character, they act on the surface of ingested lipids organized in amphiphilic droplets. In the process of emulsification, larger droplets are broken into smaller ones, thereby increasing surface to volume ratio, and, consequently, enzyme efficacy [151]. Emulsification starts with food preparation, and continues in the mouth with mastication and in the gastric and intestinal lumen with churning of the ingested food. Lipid droplets organize such that the core is composed of hydrophobic TAG, whereas amphiphilic PL, C and free fatty acids (FFA) are on the surface [152, 153]. This organization, together with some proteins and carbohydrates

Digestion of lipids exhibits a large functional redundancy [154]. It starts in the gastric lumen catalysed by the lingual and gastric lipase, together termed pre-duodenal lipase [155, 156]. The former is synthesized by acinar cells of the salivary glands and the latter by gastric chief cells. pH optimum for pre-duodenal lipase is around pH = 4, quite appropriate for the acidic gastric milieu [155, 157]. The enzyme cleaves the first ester bond in the TAG producing diacylglycerol (DAG) and an FFA [158, 159]. It is resistant to cleavage by pepsin but not by pancreatic proteases [156], and therefore functions mostly in the gastric lumen where it digests up to 15% of ingested lipids in a healthy individual. However, they might contribute to some extent to

Digestion of lipids continues in the intestinal lumen by three major enzymes secreted by pancreatic acinar cells: the (i) pancreatic, (ii) nonspecific (carboxylic ester) lipase and (iii) phospholipase A2. The pancreatic lipase is active in the presence of a colipase (which is in turn activated by trypsin), in an alkaline pH, in the presence of Ca2+ and bile salts [162, 163]. The pancreatic lipase cleaves the first and the third ester bond in TAGs yielding 2-monoacylglycerol (MAG) and FFAs [159, 164]. Phospholipase A2 acts on the second ester bond in glycerophospholipids, giving rise to lysophospholipid (LPL) and FFAs [162]. It is secreted in an inactive form and requires for its activity an alkaline pH and bile salts. The specificity of the nonspecific lipase is low, notably, and it is able to cleave cholesterol esters and MAG [162]. The quantitative contribution of the nonspecific lipase is relatively low compared with the pancreatic lipase. During infancy, the pancreatic lipase related protein 2 contributes to digestion of lipids [165].

The luminal enzyme activity outlined above, in conjunction with an ever-ongoing emulsification, results in a multi-lamellar envelope developing around the droplet, and consisting of FFAs, bile acids, C, MAG and LPLs [166–168]. The multi-lamellar envelope bursts from the droplets in the form of vesicles that are eventually transformed to mixed micelles (especially under the influence of bile salts) that then finally serve as the main vehicle for absorption of lipids. FFAs enter the enterocyte by (i) collision with the plasmalemma and crossing of the plasmalemma by the flip-flop mechanism, or (ii) by diffusion of non-ionic fatty acids or (iii) by carrier-mediated transport. The latter probably involves the fatty acid binding protein (FABP), the fatty acid transporter protein type 4 (FATP4) and CD36 [169, 170]. MAG, LPLs and C probably enter the enterocyte by means of transport proteins or by simple diffusion. Finally, the FFAs, MAG, LPLs and C are re-esterified within the enterocyte, together with apolipoproteins assembled into chylomicrons, exocytosed into the extracellular space on the basolateral side of the plasma membrane, and reach the systemic circulation via the lymphatic circulation.

stabilizes the products of emulsification.

36 Challenges in Pancreatic Pathology

digestion of lipids in the duodenum [151, 157, 160, 161].

**Figure 6.** Assimilation of lipids. (A) A schematic representation of the gastrointestinal tract. Digestion starts in the mouth and gastric lumen (red) catalysed by activity of preduodenal lipases. The exocrine pancreas secretes lipolytic enzymes (yellow) that act in the lumen of the small intestine (beige). (B) Gastric luminal digestion of lipids by preduodenal lipase. (C) Intestinal luminal digestion of lipids by pancreatic lipase, phospholipase A2 and nonspecific lipase. And (D) mixed micelles are formed in the lumen of the small intestine. These act as vehicles for absorption of FFAs, MAG, LPLs and C into enterocytes.

Exocrine pancreatic insufficiency (EPI) due to chronic pancreatitis or cystic fibrosis will eventually result in digestive malfunctioning [172]. However, because of the redundancy of the digestive processes described above, the effect is noticed rather late in the course of the disease [173]. In fact, malabsorption will not present itself until the exocrine pancreas function falls to <10% [174]. Among all the ingested nutrients, assimilation of lipids is most dependent on normal pancreas function. This seems unexpected due to a large redundancy of the lipid digestion, in fact the pre-duodenal lipase output may even increase in the EPI [175] and the pre-duodenal lipases can rescue up to 80% of fat digestion [176, 177]. However, as the destruction of the pancreas progresses, the ductal cells fail to neutralize acidic gastric juice leading to intra-intestinal acidification resulting in bile salt precipitation [175]. Bile salts are necessary for the mixed micelle formation, and this strongly hampers assimilation of lipids. Protein and carbohydrate digestion may have greater digestion potential during EPI due to the fact that the digestion is also initiated independently of pancreas, and continued by the brush-border peptidases and oligosaccharidases that are pancreas-independent. An analogue of the brushborder enzymes is missing in the digestion of lipids; therefore, it is not surprising that in EPI, lipid malabsorption is the most overwhelming problem causing many of the clinical symptoms and signs, leading to weight loss, steatorrhea, abdominal discomfort and a deficit in the lipidsoluble vitamins (A, D, E, K) [178, 179]. The digestive function can largely be rescued and nutrient malabsorption ameliorated by pancreatic enzyme replacement therapy (PERT), a therapy that involves oral administration of enzyme mixtures consisting of lipases, amylases and proteases [179–181].
