**4. Localization of pancreatic α-amylase in the small intestine**

α-Amylase synthesized in the pancreas and salivary glands is mostly secreted into the gastrointestinal tract where it digests starch. Part of the α-amylase enters the blood, one-quarter of which is excreted from the kidneys into the urine, and the remaining α-amylase is degraded (inactivated) by an unknown pathway. The α-amylase in the blood is maintained at a constant level by supply from the pancreas and salivary glands, excretion outside the body, and decomposition in the body. Therefore, the blood α-amylase activity is used for diagnosing pancreatitis and other diseases. The proportion of pancreatic α-amylase (unglycosylated 54 kDa) and saliva α-amylase (unglycosylated 54 kDa and glycosylated 57 kDa) is same when the concentration of α-amylase in human blood is examined by electrophoresis. When the pancrea is completely removed due to pancreatic cancer, the blood α-amylase activity temporarily decreases, but it returns to a normal level because the α-amylase in salivary glands increases. It is reported that fluorescently labeled α-amylase injected into rat small intestine was detected in intestinal epithelium and blood, indicating that the pancreatic α-amylase was transported into small intestine tissue (endocytosis) and blood (exocytosis) [18].

The pancreatic α-amylase-binding glycoproteins identified as Group 2 in Section 2.2 contain membrane glycoproteins that have an endocytic function. Transferrin receptor (TfR) binds to iron-bound transferrin and endocytoses iron-bound transferrin into enterocytes [19]. Similarly, aminopeptidase N, ACE2, and VLA- 2 bind to human coronavirus [20, 21], severe acute respiratory syndrome (SARS) virus [22], and matrix glycoproteins [23], respectively. Further, these ligands including viruses are able to endocytose into enterocytes. Mannose 6-phosphate receptor (Man-6-Preceptor) transports binding proteins to the lysosomal system [24]. DPP-IV does not stay in the BBM, and is transported into cells via the same pathway as aminopeptidase N and transferrin (51). This study demonstrated an endocytic pathway for α-amylase secreted into the duodenum from the pancreas [25].

#### **4.1 Endocytosis of pancreatic α-amylase into the small intestine**

Two kinds of experiments were performed using pig duodenum tissues and Caco-2 human intestinal epithelial cells that had differentiated into small

*New Insights into Metabolic Syndrome*

deficiency and remarkable fluctuations in enzymatic activity are thought to have a significant effect on starch digestion and glucose absorption. In this study, a method was established for measuring SI activity using BBM that evaluates the effects of

SGLT1 is a glycoprotein having a complex-type *N*-glycan and a sodiumdependent glucose transporter indispensable for glucose absorption in the small intestine. The effect on the SGLT1 activity means that it directly affects the blood glucose concentration. This study shows the results of examining whether pancre-

First, the timing of the addition of pancreatic α-amylase, where the effect of the α-amylase on SGLT1 activity is most frequently observed, was examined.

*-dependent glucose uptake. SGLT1 activity was assayed as Na+*

*dependent [14C]-D-glucose uptake (0.2 mM) in BBM vesicles prepared from pig duodenum. (A) Schematic illustration of the SGLT1 activity measurement system using BBM vesicles. (B) Timing of the α-amylase addition to the SGLT1 activity measurement system. T1: α-amylase was added to BBM vesicles prior to* 

*\*\*\**p *< 0.001 compared with the absence of polysaccharide by Student's* t*-test [14].*

*14C]-D-glucose; T2: α-amylase was pre-incubated with BBM vesicles 2 min before addition of [14C]-D-glucose; T3: α-amylase was pre-incubated for 15 min with [14C]-D-glucose. □: Without pancreatic α-amylase (control), ■: With pancreatic α-amylase (10 μM). (C) Effects of pancreatic α-amylase on SGLT1 acrtivity under the three conditions. (D) Dose dependency of D-glucose (D-Glc) uptake on final concentrations of added α-amylase (μM). Pancreatic α-amylase was added at T1. Results are given as means ±SE; n = 6. \**p *< 0.05,* 

*-*

The effect of pancreatic α-amylase on SI activity was investigated with and without CaCl2 and NaCl. As a comparison with α-amylase, the effects of concanavalin A (ConA), a lectin that recognizes α-mannose and α-glucose, were also measured because α-amylase shows a high affinity to α-mannose. In maltose degradation activity by SI, the α-amylase showed enhanced activity only in the presence of 5 mM CaCl2 and 0.15 M NaCl, while no effect of α-amylase was shown in the absence of CaCl2 and NaCl (**Figure 2**, left). ConA had no effect on the maltose degradation activity by SI. On the other hand, α-amylase did not affect the sucrose degradation activity, and ConA inhibited it by about 20% in the presence and absence of 5 mM CaCl2 and 0.15 M NaCl, respectively (**Figure 2**, right).

pancreatic α-amylase on SI activity as an α-glucosidase [14].

**3.2 Effects of pancreatic α-amylase on SGLT1 activity**

atic α-amylase affects SGLT1 activity.

**204**

*[*

**Figure 3.**

*Effects of pancreaitc α-amylase on Na+*

intestine-like cells by culture in Transwells for 3 weeks. In the experiment using pig duodenum tissue, fasted duodenum with no stomach contents was cut into 1-cm pieces and agitated in a pig pancreatic α-amylase solution at 37°C. After agitation for various periods of time, the tissues were fixed with formalin, and paraffin-embedded sections were prepared. The sections were immunofluorescently stained with an anti-pancreatic α-amylase antibody to clarify the localization of the α-amylase. No staining was detected in the tissue sections after incubation for 0 min. The green fluorescence with anti-pancreatic α-amylase antibody was detected at the upper end of the duodenum corresponding to the BBM in the sections incubated with α-amylase for 10 min, and α-amylase was detected in the entire duodenal tissue incubation for 30 min (**Figure 4A**). In the experiment using differentiated Caco-2 cells, AlexaFluor488-labeled human pancreatic α-amylase was added to the culture medium. After incubation for 30 min at 4°C, the α-amylase in the medium was washed out, and chased for 0–60 min at 37°C in fresh medium without α-amylase. The cells were fixed with formalin, and AlexaFluor488-labeled α-amylase was detected by confocal microscopy. Green fluorescence indicating the localization of α-amylase was detected on the cell membrane only after incubation at 4°C for 30 min. In the subsequent 37°C chase, the α-amylase was detected as punctates in the cells and had decreased at 60 min (**Figure 4B**). These results

#### **Figure 4.**

*Incorporation of pancreatic α-amylase into pig duodenum tissue (A) and differentiated human epithelial cells Caco-2 (B). (A) One-centimeter duodenum sections from fasted pigs were incubated with pig pancreatic α-amylase (10 μM) in PBS (pH 7.2) including phenylmethylsulfonyl fluoride (final 0.1 mM) at 37°C for 0, 10, or 30 min. The duodenum sections were fixed and paraffin-embedded. The paraffin sections were immunostained with rabbit anti-pancreatic α-amylase antibody as a first antibody and Alexa Fluor 488-goat anti-rabbit antibody. The green fluorescence was detected by a microscope (Olympus FSX100). (B) Caco-2 cells were seeded at 2.6 × 105 cells/cm<sup>2</sup> on polyester Transwell permeable supports (0.4-μm pores, 12-mm diameter), and cultured for about 3 weeks in minimal essential medium (DMEM, Sigma) supplemented with 20% heatinactivated (56°C, 30 min) fetal bovine serum (FBS, Gibco) and 0.1 mM non-essential amino acids (NEAA, Gibco) under a 95% air and 5% CO2 atmosphere. The cells were starved in a DMEM-NEAA medium without FBS for 18 h. After being washed with Dulbecco's Phosphate-Buffered Saline (DPBS) with Ca2+ and Mg2+, Alexa Fluor 488-human pancreatic α-amylase (20 μg/ml) in a DMEM-NEAA medium without FBS was added and incubated at 4°C for 30 min. The α-amylase was washed out, and cultured in DMEM including in 20% FBS and 1 mM NEAA at 37°C for 0–60 min. The cells were fixed, and Alexa Fluor 488-human pancreatic α-amylase was detected by confocal microscopy (ZEISS, LSM710). The nucleus was stained by 4′,6-diamidino-2-phenylindole (DAPI) [25].*

**207**

**Figure 5.**

*seeded at 2.6 × 105*

 *cells/cm<sup>2</sup>*

*Regulatory Functions of α-Amylase in the Small Intestine Other than Starch Digestion…*

intestinal epithelial cells in a time- and temperature-dependent manner.

**4.2 Endocytosis pathway of pancreatic α-amylase to lysosomes**

indicate that pancreatic α-amylase is incorporated into duodenal tissue and small

The results in **Figure 4B** showed that pancreatic α-amylase was internalized into the epithelial cells and disappeared. The cause of this disappearance is expected to be proteolysis. Among the α-amylase-binding proteins identified in Section 2.2, the membrane glycoprotein proteins classified into Group 3 are involved in protein degradation. In this study, co-localization with intracellular localization marker proteins was investigated to elucidate the endocytic pathway of the pancreatic α-amylase. Transferrin (Tf), early endosome antigen-1 (EEA1), and lysosomal-associated membrane protein 1 (LAMP1) were used as localization marker proteins of the cell membrane, early endosomes, and lysosomes, respectively, and were stained with an Alexa Fluor 594-labeled secondary antibody. Alexa Fluor 488-human pancreatic α-amylase (AF488-α-amylase) was mainly co-localized with Tf by chasing for 0–5 min at 37°C. Subsequently, the AF488 α-amylase was co-localized with EEA1 after chasing for 5–10 min, and then the α-amylase was finally co-localized with LAMP1 after a 30–60 min chase, followed by its disappearance (**Figure 5**). These results suggested that pancreatic α-amylase

*Co-localization between human pancreatic α-amylase and localization marker proteins. Caco-2 cells were* 

*DMEM supplemented with 20% heat-inactivated FBS and 0.1 mM NEAA under a 95% air and 5% CO2 atmosphere. The cells were starved in a DMEM-NEAA medium without FBS for 18 h. After being washed with DPBS containing Ca2+ and Mg2+, AF488-human pancreatic α-amylase (20 μg/ml) and/or Alexa Fluor 594 (AF594)-Tf in a DMEM-NEAA medium without FBS were added, and cells were incubated at 4°C for 30 min. The α-amylase was washed out, and cells were cultured in DMEM including 20% FBS and 1 mM NEAA at 37°C for 0–60 min. After the cells were fixed, they were stained with an anti-EEA1 antibody or anti-LAMP1 antibody as the first antibody, and followed by AF594-labeled secondary antibodies. The nucleus was stained by DAPI. The three kinds of fluorescence were detected by confocal microscopy (ZEISS, LSM710)* 

*(A) [25]. These weighted colocalizations were calculated as described previously [25] (B).*

 *on polyester Transwell permeable supports and cultured for about 3 weeks in* 

*DOI: http://dx.doi.org/10.5772/intechopen.92660*

*Regulatory Functions of α-Amylase in the Small Intestine Other than Starch Digestion… DOI: http://dx.doi.org/10.5772/intechopen.92660*

indicate that pancreatic α-amylase is incorporated into duodenal tissue and small intestinal epithelial cells in a time- and temperature-dependent manner.
