**2. Human digestive lipases**

Lipases are key enzymes responsible for digesting lipids in the digestive system. In humans, gastrointestinal lipases include pre-duodenal lipases (gastric lipase and lingual lipase) and the other members of the lipase gene family: pancreatic, hepatic, lipoprotein and endothelial lipases. The chromosomal localization of genes encoding these lipases and their tissue of origin has been described (Table 1).


\* Unknown data

Table 1. Human digestive lipases

#### **2.1 Lingual lipase**

270 New Advances in the Basic and Clinical Gastroenterology

lipoprotein and the recently described endothelial lipase. In this chapter, a short basic overview of these fat-digesting enzymes and their physiological contribution to fat digestion is first presented. Thereafter, pathophysiology of fat malabsorption resulting from exocrine pancreatic insufficiency, clinical symptoms, incidence and diagnosis of the pathology are

Standard strategies for exocrine pancreatic insufficiency management are based on oral administration of porcine derived pancreatic extracts. Unfortunately, this approach is being unsatisfactory for many reasons. Greater attention has been paid over the last decade to optimize correction of fat malabsorption and essential fatty acid deficiency in order to improve the quality of life and extend the life span of patients with severe pancreatic insufficiency. Hence, we interestingly discuss herein drawbacks of therapeutic use of currently available lipase preparations before focusing mainly on research forces joined for the development of new oral enzyme substitution approaches and future promising opportunities to treat intestinal fat malabsorption caused by exocrine pancreatic

Lipases are key enzymes responsible for digesting lipids in the digestive system. In humans, gastrointestinal lipases include pre-duodenal lipases (gastric lipase and lingual lipase) and the other members of the lipase gene family: pancreatic, hepatic, lipoprotein and endothelial lipases. The chromosomal localization of genes encoding these lipases and their tissue of

tongue

stomach

muscle

lung, kidney, placenta

Pancreatic lipase 10q26.1 Pancreas Sims & al., 1993

Hepatic lipase 15q21–q23 Liver Ameis & al., 1990

8p22 Adipose, heart, skeletal

18q21.1 Endothelial cells, liver,

**Tissue of origin References** 

Hamosh, 1990

Bodmer & al.,

Wion et al., 1987

Hirata et al., 1999

1987

described as well.

insufficiency.

Lipoprotein lipase

Endothelial lipase

\* Unknown data

Table 1. Human digestive lipases

**2. Human digestive lipases** 

origin has been described (Table 1).

**Lipase Chromosomal localization** 

**of gene** 

Lingual lipase -\* Serous glands of the

Gastric lipase 10q23.2 Fundic mucosa of the

The serous von Ebner glands of the tongue secrete lingual lipase in the saliva. Unlike rodents, lingual lipase is present in trace amounts in humans (Hamosh, 1990). Human lipase purified from lingual serous glands or gastric juice has a MW of 45 kDa to 51 kDa but tends to aggregate (MW 270-300 kDa and 500 kDa) and is highly hydrophobic (Hamosh, 1990). Lingual lipase has unique characteristics including an optimum activity at pH 4,5 – 5,4 and ability to catalyze reactions without bile salts (Hamosh & Scow, 1973). Lingual lipase breaks down short and medium chain saturated fatty acids and helps in their digestion. It has been stated that 10 to 30% of dietary fat is hydrolyzed in the stomach by lingual lipase. The enzyme uses a catalytic triad consisting of Aspartatic Acid-203 (Asp), Histidine-257 (His), and Serine-144 (Ser), to initiate the hydrolysis of a triglyceride into a diacyglyceride and a free fatty acid (Hamosh & Scow, 1973). Secreted in the buccal cavity, lingual lipase is one of the key components that make the digestion of milk fat in newborns possible. In humans lipolytic activity is present in gastric aspirates as early as 26 weeks of gestational age which is evidence enough for the fact that lingual lipase is present at birth (Hamosh, 1979). New born infants indeed secrete only low amounts of pancreatic lipase and bile salts and it has been demonstrated that pancreatic lipase alone does not readily hydrolyze a lipid emulsion as well as native milk fat globules (Miled & al., 2000).

#### **2.2 Gastric lipase**

Gastric lipase (EC 3.1.1.3) is the predominant pre-duodenal lipase in humans. The enzyme is secreted in the gastric juice by the chief cells of fundic mucosa in the stomach (Moreau & al., 1988). The pre-duodenal enzyme was purified from human gastric aspirates and its Nterminal amino-acid sequence was determined. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid unglycosylated polypeptide with a molecular weight of 43 162 Da (Bodmer & al., 1987). However, native human gastric lipase (HGL) (molecular weight 50 kDa) is a highly glycosylated protein with four potential glycosylation sites (Bodmer & al., 1987). Human gastric and rat lingual lipase share a high degree of sequence homology and have identical gene organizations (Lohse & al., 1997). Gastric lipase belongs to the α/β-hydrolase-fold family. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (backbone NH groups of Gln-154 and Leu-67) analogous to serine proteases (Roussel & al., 1999). It has an optimum pH activity around 5.4, hydrolyzes long-, medium- and short-chain triacylglycerols and do not require bile acid or colipase for optimum enzymatic activity (Denigris et al., 1985). For many years, the exact physiological contribution of gastric lipase to the overall process of lipolysis was unknown. Carrière et al. (1993a) established, for the first time, that most of the HGL secreted in the stomach was still active in the duodenum. They estimated that the gastric lipase contribution in the hydrolysis of triglycerides is about 25 %. The stereoselectivity of HGL toward triglycerides was also investigated. It was clearly demonstrated that HGL shows a stereopreference for the sn-3 position of the triglyceride (Rogalska & al., 1990).

Hence, gastric lipase, together with lingual lipase, make up 30% of lipid hydrolysis occurring during digestion in the human adult, with gastric lipase contributing the most of the two acidic lipases. In neonates, these acidic pre-duodenal lipases are much more important, they have the unique ability to initiate the degradation of maternal milk fat globules.

Emerging Approaches for the Treatment of

related protein 2 has lipase activity (Table 2).

Unknown

Phospholipids Galactolipids Esters of vitamin A

Nonspecific enzyme

Esters of cholesterol Triglycerides, diglycerides,

monoglycerides Phospholipids Ceramides

Phospholipids

Table 2. Human pancreatic enzymes involved in lipid digestion

emulsified long chain triacylglycerols (Sahasrabudhe & al., 1998).

duodenum (Nouri-Sorkhabi & al., 2000).

Abbreviations: HPL, human pancreatic lipase; TG, triglycerides

Esters of lipid-soluble vitamins

It should be stressed that adult pancreas also produces an enzyme equivalent to the colipase-dependant pancreatic lipase (CDL) called bile-salt-stimulated lipase (BSSL) or carboxyl ester lipase (EC 3.1.1.1) (Hui & Howles, 2002). Originally discovered in milk of humans and various other primates (Swan & al., 1992), BSSL participates to the intestinal digestion of dietary lipids (Table 2). While colipase-dependent pancreatic lipase facilitates the uptake of fatty acids, bile-salt-stimulated lipase facilitates the uptake of free cholesterol from the intestinal lumen (Sahasrabudhe & al., 1998). A distinguishing feature of this 722 amino acid native protein is that it requires primary bile salts for the hydrolysis of

The exocrine pancreas secretes another group of phospholipid-hydrolyzing enzymes including phospholipase A1 (EC 3.1.1.32), and phospholipase A2 (EC 3.1.1.4). These enzymes are secreted in their zymogen form and activated by trypsin on entering the

Pancreatic lipase related protein 1

Pancreatic lipase related protein 2

Carboxyl ester lipase

Phospholipase A2

(PLRP1)

(PLRP2)

Fat Malabsorption due to Exocrine Pancreatic Insufficiency 273

The lipase gene family includes also two other pancreatic proteins: pancreatic lipase related proteins 1 and 2, with strong nucleotide and amino acid sequence homology to pancreatic triglyceride lipase. All three proteins have virtually identical three-dimensional structures (Lowe, 2000). Of the pancreatic triglyceride lipase homologues, only pancreatic lipase

**Enzyme Name Substrate specificity References**  Pancreatic lipase Triglycerides (lipid-droplet) Thirstrup & al., 1994

> Crenon & al., 1998 Berton & al., 2009

Berton & al., 2009

Thirstrup & al., 1994 Sias & al., 2004 Reboul & al., 2006

Hui & Howles , 2002

Verheij & al., 1983

Inhibitory effect of HPL (regulatory effect of TG digestion in the duodenum?)

Broad range of substrate specificity Triglycerides (milk lipid-droplet) Synergistic effect of HPL (regulatory effect of TG digestion in the duodenum?)

### **2.3 Pancreatic lipases**

A limitation of acidic lipases is that they remove only one fatty acid from each triacylglycerol. The free fatty acid can readily cross the epithelial membrane lining the gastrointestinal tract, but the diacylglycerol cannot be transported across. Hence, hydrolysis of dietary triacylglycerols by both gastric and pancreatic lipase is essential for their absorption by enterocytes. Human pancreatic lipase (HPL) (EC 3.1.1.3) is produced by the pancreatic acinar cells. The lipase is located into the zymogen granules, together with many other enzymes and secreted into the intestinal lumen together with the bile (Miled & al., 2000). Contrary to most of the pancreatic enzymes which are secreted as proenzymes and further activated by proteolytic cleavage in the small intestines, HPL is directly secreted as an active enzyme. Purified from pancreatic juice, the protein showed to have a molecular weight of 48 kDa (De Caro & al., 1977) and has been characterized as a glycoprotein consisting of 449 amino acid polypeptide (Lowe & al., 1989). The resolution of the HPL 3D structure (Fig.1) revealed the presence of a catalytic triad (Ser152-Asp176 –His263) similar to that found in other serine hydrolases, Ser l52 being part of the G-X-S-X-G consensus sequence (Lowe & al., 1989). Pancreatic lipase acts maximally around pH 8-9 (Winkler et al., 1990) and was found to be poorly stereoselective (Rogalska & al., 1990). Unlike pre-duodenal lipases, pancreatic lipase requires colipase—a pancreatic protein—as cofactor for its enzymatic activity (Fig.1). Colipase relieves phosphatidyl choline-mediated inhibition of the interfacial lipase–substrate complex, helps anchor the lipase to the surface and stabilizes it in the 'open', active conformation (Brockman, 2000; Lowe, 1997).

Fig. 1. 3-D Structure of the HPL-procolipase complex in the closed conformation (E) and in the open conformation (E\*). These two diagrams show the conformational changes in the lid, the β-5 loop and the colipase during interfacial activation (Adapted from Miled et al., 2000 as cited in van Tilbeurgh et al., 1992; 1993).

A limitation of acidic lipases is that they remove only one fatty acid from each triacylglycerol. The free fatty acid can readily cross the epithelial membrane lining the gastrointestinal tract, but the diacylglycerol cannot be transported across. Hence, hydrolysis of dietary triacylglycerols by both gastric and pancreatic lipase is essential for their absorption by enterocytes. Human pancreatic lipase (HPL) (EC 3.1.1.3) is produced by the pancreatic acinar cells. The lipase is located into the zymogen granules, together with many other enzymes and secreted into the intestinal lumen together with the bile (Miled & al., 2000). Contrary to most of the pancreatic enzymes which are secreted as proenzymes and further activated by proteolytic cleavage in the small intestines, HPL is directly secreted as an active enzyme. Purified from pancreatic juice, the protein showed to have a molecular weight of 48 kDa (De Caro & al., 1977) and has been characterized as a glycoprotein consisting of 449 amino acid polypeptide (Lowe & al., 1989). The resolution of the HPL 3D structure (Fig.1) revealed the presence of a catalytic triad (Ser152-Asp176 –His263) similar to that found in other serine hydrolases, Ser l52 being part of the G-X-S-X-G consensus sequence (Lowe & al., 1989). Pancreatic lipase acts maximally around pH 8-9 (Winkler et al., 1990) and was found to be poorly stereoselective (Rogalska & al., 1990). Unlike pre-duodenal lipases, pancreatic lipase requires colipase—a pancreatic protein—as cofactor for its enzymatic activity (Fig.1). Colipase relieves phosphatidyl choline-mediated inhibition of the interfacial lipase–substrate complex, helps anchor the lipase to the surface and stabilizes it in the

Fig. 1. 3-D Structure of the HPL-procolipase complex in the closed conformation (E) and in the open conformation (E\*). These two diagrams show the conformational changes in the lid, the β-5 loop and the colipase during interfacial activation (Adapted from Miled et al.,

'open', active conformation (Brockman, 2000; Lowe, 1997).

2000 as cited in van Tilbeurgh et al., 1992; 1993).

**2.3 Pancreatic lipases** 

The lipase gene family includes also two other pancreatic proteins: pancreatic lipase related proteins 1 and 2, with strong nucleotide and amino acid sequence homology to pancreatic triglyceride lipase. All three proteins have virtually identical three-dimensional structures (Lowe, 2000). Of the pancreatic triglyceride lipase homologues, only pancreatic lipase related protein 2 has lipase activity (Table 2).


Abbreviations: HPL, human pancreatic lipase; TG, triglycerides

Table 2. Human pancreatic enzymes involved in lipid digestion

It should be stressed that adult pancreas also produces an enzyme equivalent to the colipase-dependant pancreatic lipase (CDL) called bile-salt-stimulated lipase (BSSL) or carboxyl ester lipase (EC 3.1.1.1) (Hui & Howles, 2002). Originally discovered in milk of humans and various other primates (Swan & al., 1992), BSSL participates to the intestinal digestion of dietary lipids (Table 2). While colipase-dependent pancreatic lipase facilitates the uptake of fatty acids, bile-salt-stimulated lipase facilitates the uptake of free cholesterol from the intestinal lumen (Sahasrabudhe & al., 1998). A distinguishing feature of this 722 amino acid native protein is that it requires primary bile salts for the hydrolysis of emulsified long chain triacylglycerols (Sahasrabudhe & al., 1998).

The exocrine pancreas secretes another group of phospholipid-hydrolyzing enzymes including phospholipase A1 (EC 3.1.1.32), and phospholipase A2 (EC 3.1.1.4). These enzymes are secreted in their zymogen form and activated by trypsin on entering the duodenum (Nouri-Sorkhabi & al., 2000).

Emerging Approaches for the Treatment of

activating the enzyme (Mukherjee, 2003).

**2.6 Endothelial lipase** 

**healthy state** 

of triglycerides (Fig.2).

Fat Malabsorption due to Exocrine Pancreatic Insufficiency 275

homodimer, LPL has the dual function of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. Through catalysis, triacylglycerol present in very low-density lipoprotein (VLDL) and chylomicron particles is converted to triglyceride-poor intermediate-density lipoprotein (IDL) and chylomicron remnants, respectively (Mukherjee, 2003). Apolipoprotein CII (ApoCII) present on VLDL particles is the co-factor required for

Endothelial lipase (EC 3.1.1.3) which was firstly characterized in 1999 was also added to the lipase gene family (Jaye & al., 1999). Mature endothelial lipase is a 68 kDa glycoprotein with five potential N-linked glycosylation sites (Yasuda & al., 2010). It has 44% primary sequence homology with lipoprotein lipase, 41% with hepatic lipase and 27% with pancreatic lipase (Choi & al., 2002). The enzyme is secreted by endothelial cells from various tissues like lung, liver, kidney and placenta. However, heart and skeletal muscles do not express endothelial lipase (Jaye & al., 1999). Endothelial lipase differs from the other enzymes of the lipase gene family in the sequence of the 'lid' domain. Its 19-residue 'lid' region is 3 residues shorter and less amphipathic than 'lid' region of lipoprotein or hepatic lipase indicating a different enzymatic function (Jaye & al., 1999). Indeed, unlike lipoprotein or hepatic lipases that have triacylglycerol lipase activity, endothelial lipase has primarily a phospholipase A1 activity. It was suggested that endothelial lipase plays a physiologic role in HDL metabolism probably by catalyzing hydrolysis of HDL phospholipids thereby facilitating a direct HDL receptormediated uptake (Cohen, 2003). Endothelial lipase may also facilitate the uptake of apolipoprotein B-containing remnant lipoprotein. As the placental tissue abundantly expresses endothelial lipase, it may also have a role in the development of fetus (Choi & al., 2002).

**3. Human dietary lipid digestion process and regulation of pancreatic fluid in** 

Protein digestion begins in the stomach with the concomitant action of hydrochloric acid and pepsin, continues with pancreatic proteases in the duodenum, and finishes with numerous brush border peptidases located all over the small intestine (Fieker & al., 2011 as cited in Alpers, 1994). Starch digestion begins in the mouth with salivary amylase, continues with pancreatic amylase, and ends with several intestinal brush border oligosaccharidases (Fieker & al., 2011 as cited in Alpers, 1994). In contrast, the majority of lipid digestion and absorption occurs between the pylorus and the ligament of Treitz. Prior to this step, 5% to 40% of the dietary triglyceride acyl chains are released in the stomach by gastric lipase (Armand & al., 1994, 1996, 1999; Carrière & al., 1993b ; Hamosh, 1990) which continues its action in the duodenum together with pancreatic lipase until these enzymes are degraded by pancreatic proteases. Although a pH of 8 to 9 appears to be optimal for pancreatic lipase activity *in vitro*, bile salts allow the enzyme to work efficiently at a pH of 6 to 6.5 *in vivo* (Borgstrom, 1964; Carrière & al., 2005). HPL is responsible for the hydrolysis of 40% to 70%

The pancreatic lipase-related 2 protein (hPLRP2), with a broader substrate specificity, hydrolyzes milk triglycerides (Berton & al., 2009) phospholipids (Jayne & al., 2002; Lowe, 2002; Thirstrup & al., 1994) galactolipids (Sias & al., 2004) and esters of lipid-soluble vitamins (Reboul & al., 2006). Carboxyl ester lipase (also called bile salt-stimulated lipase,

PLA1 catalyzes the hydrolysis of fatty acids exclusively at the sn-1 position of phospholipids. A free fatty acid (FFA) and a lysophospholipid (lysoPL) are the products of this reaction. However, this class of phospholipase is not well understood, and no crystal structures exist. The assignment of a function for this pancreatic enzyme has yet to be firmly established (Richmond & Smith, 2011).

In intraluminal digestion, phospholipase A2 is primarily responsible for hydrolyzing phosphatidyl-choline to 2-lysophophatidyl-choline. This reaction is important in triglyceride digestion as the amphipathic phosphatidyl-choline, in a manner similar to bile salts, will adsorb to the surface of the lipid droplets, preventing contact between the lipase-colipase complex and its lipid substrate. Hydrolysis of phosphatidyl-choline by phospholipase 2 will allow desorption of lysophosphatidyl-choline, which is water soluble. The subsequent mucosal absorption of lysophosphatidyl-choline is important in the generation of enterocyte phospholipids and lipoproteins and, thus, chylomicron formation (Nouri-Sorkhabi & al., 2000).
