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

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

#### **Figure 5.**

*New Insights into Metabolic Syndrome*

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

*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* 

*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* 

 *on polyester Transwell permeable supports (0.4-μm pores, 12-mm diameter),* 

**206**

**Figure 4.**

*were seeded at 2.6 × 105*

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

*4′,6-diamidino-2-phenylindole (DAPI) [25].*

*Co-localization between human pancreatic α-amylase and localization marker proteins. Caco-2 cells were seeded at 2.6 × 105 cells/cm<sup>2</sup> on polyester Transwell permeable supports and cultured for about 3 weeks in 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).*

binds to the cell membrane and is transported into lysosomes though early endosomes and the α-amylase undergo degradation in the lysosome. The disappearance of AF488-amylases endocytosed into the cells was suppressed by chloroquine, which is an inhibitor of lysosome proteolysis (data not shown) [25]. In another experiment using pig duodenum tissue sections, α-amylase in the duodenum was well co-localized with LAMP1, and its degraded fragments were detected (data not shown) [25]. These results indicate that pancreatic α-amylase internalized by the endocytic pathway is undergoing proteolysis in the lysosome. Degraded α-amylase may be used as a source for rapid turnover in duodenal epithelial cells.
