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

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 pancreatic α-amylase affects SGLT1 activity.

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.

#### **Figure 3.**

*Effects of pancreaitc α-amylase on Na+ -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 [ 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, \*\*\**p *< 0.001 compared with the absence of polysaccharide by Student's* t*-test [14].*

**205**

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

T1: Pancreatic α-amylase was added just before the [14C]-D-glucose substrate solution without preincubation. T2: The α-amylase was pre-incubated with BBM vesicle solution at 37°C for 2 min. T3: The α-amylase was pre-incubated with [14C]-Dglucose substrate solution at 37°C for 15 min (**Figure 3A** and **B**). Inhibition of SGLT1 acitivity by pancreatic α-amylase was shown at all tested additions of the α-amylase, and T1 was the most efficient for SGLT1 inhibition (**Figure 3C**). The concentration-dependent effects of α-amylase on SGLT1 activity were examined under the T1 condition. SGLT1 activity was reduced from 5 μM of α-amylase, and inhibited to 34% at 10 μM and 10% at 20 μM (**Figure 3D**). The 50% inhibitory

Pancreatic α-amylase concentrations in pancreatic juice have been reported to be 4.2 mg/ml in pigs and 5–16 mg/ml in cows [15, 16]. Humans secrete 1–3 L of pancreatic juice containing several to several tens of grams of protein per day. Therefore, pancreatic α-amylase is probably present in the order of mg/ml because the total concentration of α-amylase in pancreatic juice protein is 26.5% [17]. In this study, it was found that SGLT1 activity was inhibited by pancreatic α-amylase at more than

**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

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

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

concentration (IC50) value was 8.1 μM.

tissue (endocytosis) and blood (exocytosis) [18].

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

10 μM (≒ 0.56 mg/ml).

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

T1: Pancreatic α-amylase was added just before the [14C]-D-glucose substrate solution without preincubation. T2: The α-amylase was pre-incubated with BBM vesicle solution at 37°C for 2 min. T3: The α-amylase was pre-incubated with [14C]-Dglucose substrate solution at 37°C for 15 min (**Figure 3A** and **B**). Inhibition of SGLT1 acitivity by pancreatic α-amylase was shown at all tested additions of the α-amylase, and T1 was the most efficient for SGLT1 inhibition (**Figure 3C**). The concentration-dependent effects of α-amylase on SGLT1 activity were examined under the T1 condition. SGLT1 activity was reduced from 5 μM of α-amylase, and inhibited to 34% at 10 μM and 10% at 20 μM (**Figure 3D**). The 50% inhibitory concentration (IC50) value was 8.1 μM.

Pancreatic α-amylase concentrations in pancreatic juice have been reported to be 4.2 mg/ml in pigs and 5–16 mg/ml in cows [15, 16]. Humans secrete 1–3 L of pancreatic juice containing several to several tens of grams of protein per day. Therefore, pancreatic α-amylase is probably present in the order of mg/ml because the total concentration of α-amylase in pancreatic juice protein is 26.5% [17]. In this study, it was found that SGLT1 activity was inhibited by pancreatic α-amylase at more than 10 μM (≒ 0.56 mg/ml).
