**5. Expression of endogenous α-amylase in the human duodenum**

α-Amylase is abundantly expressed in the pancreas and salivary glands, and has been detected in liver [26], thyroid [27], parotid gland [28], white blood cells [29], lung carcinoma tissues [30], and brain [3] in humans. There are five isoforms of α-amylase including three salivary α-amylases (isoforms *AMY1A*, *AMY1B*, and *AMY1C*) and two pancreatic α-amylases (isoforms *AMY2A* and *AMY 2B*) [31]. The α-amylases in liver, white blood cells, and lung carcinoma tissues are encoded by *AMY2B* [2, 29, 30]. The brain amylases are encoded by *AMY1A* and *AMY2B* [3]. The parotid gland amylase is encoded by *AMY1C* [28]. It is reported that α-amylase activity is detected in small intestinal tissues [32]. It is not clear whether this α-amylase is an exogenous one that endocytoses pancreatic α-amylase into small intestinal tissues or an endogenous α-amylase that is expressed in the small intestine itself. An endogenous α-amylase was stained in differentiated Caco-2 cells by immunostaining with an anti-α-amylase antibody during the experiments on internalization using fluorescence-labeled α-amylase in the cells described in Section 4. In this study, we demonstrated the expression of endogenous α-amylase in human duodenal epithelial cells and identified the isoform of the α-amylase expressed in the duodenal epithelial cells. Furthermore, the biological significances of α-amylase expression in the duodenum are shown [33].

#### **Figure 7.**

*New Insights into Metabolic Syndrome*

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.

Based on the SGLT1 inhibition of pancreatic α-amylase described in Section 3.2, the biological significance of endocytosis of the pancreatic α-amylase in Sections 4.1 and 4.2 is discussed as follows. **Figure 6** shows a hypothetical scheme of the biological function of α-amylase internalization [25]. (i) Within 30 min after food intake, pancreatic α-amylase has begun to be secreted from the pancreas into the duodenum, but its amount is not high. Therefore, the α-amylase on the surface of the duodenal BBM interacts with the SI on the BBM to promote starch digestion by both the α-amylase and SI. (ii) Approximately 30 min after food intake, the concentration of pancreatic α-amylase in the small intestinal lumen reaches a maximum, and the α-amylase binds to the glycoproteins in the BBM. At this time, the glucose uptake of SGLT1 is inhibited by the α-amylase, whereby rapid glucose uptake is suppressed, and postprandial hyperglycemia is corrected. (iii) At 30–60 min after food intake, the α-amylase bound to the BBM is internalized into early endosomes in the epithelial cells.

**4.3 Biological significances of endocytosis of pancreatic α-amylase**

**208**

**Figure 6.**

*A hypothetical schematic of the biological function of pancreatic α-amylase internalization.*

*Expression of α-amylase in human tissues. A reaction mixture was prepared by mixing Human MTC panel I, II or Digestive system MTC panel (Clontech) as a cDNA (5 μl), Power SYBER Green PCR Master mix (6.25 μl), 50 μM forward and reverse primers for AMY2B (0.2 μl each), and putting it in the 96-well reaction plate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard. The sequences of primer sets for AMY2B and GAPDH were described previously [33]. Normalized data by GAPDH are shown with liver expression as 1.*

## **5.1 Expression of** *AMY2B* **in human duodenum tissues**

The mRNA expression of α-amylase in human tissues was measured by realtime PCR using human Multiple Tissue cDNA (MTC) Panels. The α-amylase was shown to be expressed most in the pancreas and then in the duodenum (**Figure 7**). The results mean that the α-amylase detected in the duodenum consists of both the endogenous α-amylase expressed in the duodenum and the exogenous α-amylase expressed by the pancreas and internalized into the duodenum. For further investigation, mRNA levels, protein levels, enzyme activities, and localization of the α-amylases were clarified using differentiated Caco-2 cells [33]. Caco-2 cells were originally human intestinal epithelial cells derived from the colon, but are known to gradually develop small intestine-like properties when differentiated by longterm culture for about 3 weeks. Therefore, we focused on the change in α-amylase expression in Caco-2 cells during differentiation into small intestine-like cells. The expression of α-amylase in Caco-2 cells increased with differentiation into small intestine-like cells in its mRNA level, protein level, enzyme activity, and immunostaining (**Figure 8**). Activity of α-amylase was detected in the cell extract but not in the culture medium, suggesting that the α-amylase was not secreted by the cells

#### **Figure 8.**

*Expression of α-amylase in differentiated human epithelial cells. Caco-2 cells were seeded at 5 × 104 cells/cm<sup>2</sup> in the wells and cultured for 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 washed with DPBS and were prepared for each measurement as described previously [33]. (A) mRNA expression of α-amylase in the cells was measured by quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems). \**p *< 0.05, \*\**p *< 0.01, \*\*\**p *< 0.001 vs. Day 2 by one-way ANOVA with Dunnett's posthoc test. (B) Protein levels of α-amylase in the cell extract were detected by Western blotting using anti-human pancreatic α-amylase antibody (HPA IgGs) as a first antibody and HRP-labeled secondary antibodies. The staining was developed by 3,3′-diaminobenzidine (DAB) including H2O2. (C) Specific enzymatic activities of α-amylase in the culture medium and cell extract. \**p *< 0.05, \*\**p *< 0.01 vs. medium by paired* t*-test (D) Immunofluorescence staining of the permeabilized cells with anti-HPA IgGs as a first antibody and AF488-labeled secondary antibodies. The nucleus was stained by DAPI. The fluorescence was detected by confocal microscopy (ZEISS, LSM710). The lower right is an enlarged view [33].*

**211**

tration (**Figure 9B**).

**6. Conclusion**

**Figure 9.**

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

 *(A) or 1 × 105*

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

*\*\**p *< 0.01, \*\*\**p *< 0.001, one-way ANOVA with Tukey's posthoc test [33].*

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

(**Figure 8C**). Immunostaining showed that the α-amylase in the cytoplasm was localized in a dotted pattern (**Figure 8D**). The isotype of the α-amylase expressed in differentiated Caco-2 cells was identified as pancreatic type *AMY2B* by a combina-

*Effects of α-amylase suppression by siRNA on cell differentiation and proliferation. Nonspecific siRNA as a control or the siRNA targeting α-amylase was transfected to Caco-2 cells and these cells were seeded at 2 × 105*

*with 20% heat-inactivated FBS and 0.1 mM NEAA under a 95% air and 5% CO2 atmosphere. (A) The cells were washed with DPBS and were prepared for mRNA expression as described previously [33]. mRNA expressions of α-amylase differentiation marker proteins (SI, DPP-4, E-cadherin, villin) in the cells were measured by quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems). (B) The cells in the 96-well plates were stained with MTT, and the cell viability was measured by an MTT assay. Mock, the cells transfected with the transfection reagent only without siRNA. Data are shown as mean ± SE, \**p *< 0.05,* 

 *(B) in the wells. The cells were cultured for 3 days in DMEM supplemented* 

α-Amylase has been shown to be expressed by tissues other than the pancreas and salivary glands, but the biological significance has not been elucidated. The increased α-amylase expression in obese mouse liver suggests that liver α-amylase may be a biomarker for obesity [34]. It has been hypothesized that α-amylase expressed in the brain may be an energy source in Alzheimer's disease [3]. Here, it was shown that the expression of α-amylase by Caco-2 cells was suppressed by RNA interference (RNAi), and affected cell proliferation and differentiation [33]. The expression of α-amylase was suppressed to about 30% by siRNA (small interfering RNA), and four kinds of cell differentiation markers were quantified by real-time PCR. All four differentiation markers were reduced in cells transfected with α-amylase siRNA compared to cells transfected with control siRNA (**Figure 9A**). This result indicates that α-amylase expression is required for cell differentiation. Furthermore, it was shown that the cell proliferation of cells transfected with α-amylase siRNA was dramatically reduced depending on the cell-seeding concen-

This study showed new functions of both exogenous and endogenous pancreatic α-amylase other than starch digestion in the small intestine. Exogenous α-amylase synthesized by the pancreas and secreted into the small intestinal tract enhances α-glucosidase activity and inhibits glucose uptake by SGLT1 in the small

tion of PCR and restriction enzyme treatment (data not shown) [33].

**5.2 Biological significances of α-amylase expressed in the duodenum**

*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*

#### **Figure 9.**

*New Insights into Metabolic Syndrome*

**5.1 Expression of** *AMY2B* **in human duodenum tissues**

The mRNA expression of α-amylase in human tissues was measured by realtime PCR using human Multiple Tissue cDNA (MTC) Panels. The α-amylase was shown to be expressed most in the pancreas and then in the duodenum (**Figure 7**). The results mean that the α-amylase detected in the duodenum consists of both the endogenous α-amylase expressed in the duodenum and the exogenous α-amylase expressed by the pancreas and internalized into the duodenum. For further investigation, mRNA levels, protein levels, enzyme activities, and localization of the α-amylases were clarified using differentiated Caco-2 cells [33]. Caco-2 cells were originally human intestinal epithelial cells derived from the colon, but are known to gradually develop small intestine-like properties when differentiated by longterm culture for about 3 weeks. Therefore, we focused on the change in α-amylase expression in Caco-2 cells during differentiation into small intestine-like cells. The expression of α-amylase in Caco-2 cells increased with differentiation into small intestine-like cells in its mRNA level, protein level, enzyme activity, and immunostaining (**Figure 8**). Activity of α-amylase was detected in the cell extract but not in the culture medium, suggesting that the α-amylase was not secreted by the cells

**210**

*view [33].*

**Figure 8.**

*Expression of α-amylase in differentiated human epithelial cells. Caco-2 cells were seeded at 5 × 104*

*in the wells and cultured for 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 washed with DPBS and were prepared for each measurement as described previously [33]. (A) mRNA expression of α-amylase in the cells was measured by quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems). \**p *< 0.05, \*\**p *< 0.01, \*\*\**p *< 0.001 vs. Day 2 by one-way ANOVA with Dunnett's posthoc test. (B) Protein levels of α-amylase in the cell extract were detected by Western blotting using anti-human pancreatic α-amylase antibody (HPA IgGs) as a first antibody and HRP-labeled secondary antibodies. The staining was developed by 3,3′-diaminobenzidine (DAB) including H2O2. (C) Specific enzymatic activities of α-amylase in the culture medium and cell extract. \**p *< 0.05, \*\**p *< 0.01 vs. medium by paired* t*-test (D) Immunofluorescence staining of the permeabilized cells with anti-HPA IgGs as a first antibody and AF488-labeled secondary antibodies. The nucleus was stained by DAPI. The fluorescence was detected by confocal microscopy (ZEISS, LSM710). The lower right is an enlarged* 

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

*Effects of α-amylase suppression by siRNA on cell differentiation and proliferation. Nonspecific siRNA as a control or the siRNA targeting α-amylase was transfected to Caco-2 cells and these cells were seeded at 2 × 105 cells/cm<sup>2</sup> (A) or 1 × 105 cells/cm<sup>2</sup> (B) in the wells. The cells were cultured for 3 days in DMEM supplemented with 20% heat-inactivated FBS and 0.1 mM NEAA under a 95% air and 5% CO2 atmosphere. (A) The cells were washed with DPBS and were prepared for mRNA expression as described previously [33]. mRNA expressions of α-amylase differentiation marker proteins (SI, DPP-4, E-cadherin, villin) in the cells were measured by quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems). (B) The cells in the 96-well plates were stained with MTT, and the cell viability was measured by an MTT assay. Mock, the cells transfected with the transfection reagent only without siRNA. Data are shown as mean ± SE, \**p *< 0.05, \*\**p *< 0.01, \*\*\**p *< 0.001, one-way ANOVA with Tukey's posthoc test [33].*

(**Figure 8C**). Immunostaining showed that the α-amylase in the cytoplasm was localized in a dotted pattern (**Figure 8D**). The isotype of the α-amylase expressed in differentiated Caco-2 cells was identified as pancreatic type *AMY2B* by a combination of PCR and restriction enzyme treatment (data not shown) [33].
