Type 1 Diabetes Accelerates Small Bowel Transit By Enhancing Store-operated Ca²⁺ Signal Of Small Intestinal Smooth Muscle

BackgroundDiabetic enteropathy is a common complication of type 1 diabetes (T1D), with a usual symptom of diarrhea, which may be related to dysfunction of intestinal motility. Smooth intestinal muscle tissue plays a very important role in maintaining the physiological function of intestinal constraction, and Ca2+is the important "second messenger" for smooth muscle cell contraction. Store-operated Ca2+entry (SOCE) is a ubiquitous mechanism of Ca2+influx. Orail and stromal interaction molecule 1 (STTM1) is the main component of the SOCC channel. Orail is expressed at the cell surface, as the pore subunit. SOCE is initiated by STIM1, which sense ER Ca2+levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated Orai1. Many physiological functions will be changed with hyperglycemia induced by Diabetes. Therefore, we speculate that the gastrointestinal (GI) disorder caused by diabetes may be associated with SOCE.Objective1. To investigate the effect of HG (high glucose) environment on SOCE in MUS-M1 cells.2. Study the role of Orail and STIM1 in small intestine contraction and gastrointestinal transit in diabetic mice.Method1. [Ca2+] i measurmentSOCE was activated by Ca2+depletion evoked by agonists in MUS-M1 cultured with HG and NG. Use Calcium imaging fluorescence microscope system to detect changes of intracellular Ca2+concentration.2. Western BlotWestern Blot was used to detect the expression levels of Orail and STIM1 proteins in NG-and HG-cultured MUS-M1 cells.3. siRNA transient transfectionSpecific siRNAs against mice STIM1, Orail or scramble control siRNA were transiently transfected into MUS-M1 cells. Cells were harvested for the following experiments at 36 h after siRNA transfection.4. Induction of type 1 diabetes5-week-old male Kunming mice were randomly divided into STZ group and control group. In the STZ group were administered multiple, low-dose STZ (40 mg/kg) for 5 consecutive days to induce type 1 diabetes. Mice in the control group received an injection of the acetate buffer. Mice fasting plasma glucose levels were higher than 11.1 mmol/L were selected to use in our study.5. The expression of Orail STIM1 and BKCa protein in small intestinal smooth muscle tissue of diabetic miceWestern Blot was used to detect the expression levels of STIM1, Orai1 and BKCa in the small intestinal smooth muscle tissue of diabeties mice.6. Preparation of isolated intestine strip segment and tension measurementIntestine was quickly dissociated fand placed in Krebs-Henseleit solution, connected to isometric force transducer linked with intregrated amplifier and recorder. After then, we used Ca2+ after carbachol (CCh) to induce tension response curves in 0Ca2+ Krebs. The isometric tension was recorded by a data acquisition and analysis system (BL-420S).7. Intestinal transitCharcoal meal marker was adminstered orally to assess upper gastrointestinal transit. The distance travelled by the marker was measured in centimetre and expressed as percentage of the total length of the small intestine from pylorus to terminal ileum.8. Co-immunoprecipitationUse Co-immunoprecipitation to examine the change of the association between Orail and BKCa.Result(1) SOCE in MUS-M1 was enhanced by prolonged HG treatment.(2) The expression levels of Orail and STIM1 were significantly enhanced in the HG-cultured cells at 3 and 7 days.(3) The decrease of Orail and STIM1 expression attenuate the intensity of SOCE in HG and NG-cultured MUS-M1 cells.(4) The expression levels of Orail and STIM1 were significantly increased in the small intestinal smooth muscle of diabetic mice.(5) SOCE-mediated small intestine segment contraction and gastrointestinal transit was significantly increased in diabetic mice compared control mice.(6) Orail and STIM1 specific siRNAs transfection significantly decreased the small intestine segment contraction and gastrointestinal transit of diabetic mice.(7) Interaction of Orail and BKCa became weaken in the small intestinal smooth muscle of diabetic mice.Conclusion1. HG environment enhanced SOCE of MUS-M1 cells. Change of Orail and STIM1 protein expression affect the intensity of SOCE in MUS-M1.2. Orail and STIM1 play important roles in small intestine segment contraction and gastrointestinal transit...