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Application of FMO3 as target in screening of drugs or models for preventing and treating pancreas islet function damage

A technology of pancreatic islets and drugs, applied in the field of biomedicine, can solve problems such as unreported relationship, and achieve the effect of reducing β-cell dysfunction and improving insulin secretion deficiency.

Pending Publication Date: 2022-03-01
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In human clinical samples, high plasma TMAO levels are closely related to type 2 diabetes, and diabetic mice also have higher TMAO levels, but the relationship between TMAO and FMO3 and glucose-stimulated insulin secretion has not been reported

Method used

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  • Application of FMO3 as target in screening of drugs or models for preventing and treating pancreas islet function damage
  • Application of FMO3 as target in screening of drugs or models for preventing and treating pancreas islet function damage
  • Application of FMO3 as target in screening of drugs or models for preventing and treating pancreas islet function damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Determination of TMAO concentration in plasma

[0066] 1.1 Test conditions

[0067] 1.1.1 Standard curve establishment

[0068] Since TMAO may be contained in normal mouse plasma, the references used purified water to prepare the standard curve. TMAO was dissolved in purified water and diluted into working solutions with concentrations of 0.2 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM. Add 10 μL of TMAO working solution with different concentrations to 40 μL of acetonitrile containing internal standard (d9TMAO, 50 ng / mL), and after mixing, take 3 μL of the supernatant for LC / MS / MS analysis.

[0069] 1.1.2 Plasma sample processing

[0070] Add 40 μL of acetonitrile containing internal standard (d9TMAO, 50 ng / mL) to 10 μL of plasma sample, vortex and centrifuge (14000 rpm×5 min) twice, and take 5 μL of supernatant for LC / MS / MS analysis. The samples above the standard line were diluted 100 times for determination.

[0071] 1.1.3 LC / MS conditions

[0072]...

Embodiment 2

[0086] Confirmation that TMAO affects insulin secretion function at the cellular level

[0087] 1×10 per hole 5 Min6 cells were plated in a 24-well plate, and after 2 days, different concentrations (100nM, 1μM, 10μM) of TMAO were added to treat the islet β cell line Min6 for 1h, 6h, 12h, and 18h, and GSIS was performed; low-sugar 2.8mM glucose KRHB buffer was washed once Cells were starved for 1 hour in low-sugar 2.8mM glucose KRHB buffer, stimulated in high-sugar 16.8mM glucose KRHB buffer for 1 hour, and the supernatant was collected to measure insulin levels with the insulin ELISA kit. Determine the onset concentration and onset time of TMAO.

[0088] Min6 was treated with TMAO for 15h, cell viability was detected by CCK8 method, 2.5×10 per well 5 Min6 cells were plated in a 96-well plate, treated with 100 nM and 10 μM TMAO for 15 hours after 2 days, and 10 μl of CCK8 solution was added to each well, at 37°C, 5% CO 2 Incubate in the incubator for 1h, measure OD 450 .

...

Embodiment 3

[0092] Confirmation that TMAO produced by FMO3 affects insulin secretion function at the animal level

[0093] Choline in the diet is an important source of TMAO. After choline is converted into TMA in the body, it is oxidized to TMAO by FMO3. Control diet (0.08% choline) and high choline diet (1% choline) fed C57BL / 6J mice, and LC / MS determined the level of TMAO in mouse plasma to confirm that high choline diet induced the increase of TMAO level; weighed ; food intake was recorded; glucose tolerance of mice was detected by IPGTT; blood was collected by intraperitoneal injection of glucose for 2 minutes and 5 minutes, insulin level was measured by insulin ELISA kit, C-peptide level was measured by C-peptideELISA kit; insulin sensitivity of mice was detected by ITT; high glucose clamp Measure blood sugar, glucose input rate and insulin secretion level, and evaluate the function of pancreatic beta cells.

[0094] The mouse primary islets were isolated for GSIS to detect the ins...

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Abstract

The invention belongs to the technical field of medicine, and relates to application of synthase FMO3 of intestinal flora metabolite TMAO as a target in screening of drugs or models for preventing and treating pancreas islet function damage. Specifically, the invention discloses application of FMO3 as a target in screening of drugs or biological agents for preventing and treating diseases caused by pancreas islet function impairment and application of FMO3 in preparation of drugs or biological agent models for screening of diseases caused by pancreas islet function impairment. According to the invention, FMO3 is taken as a target and is applied to screening of drugs for promoting glucose-stimulated insulin secretion. The product of FMO3 is TMAO, and a large number of tests prove that TMAO obviously inhibits insulin secretion stimulated by mouse pancreatic beta cell Min6 glucose, knocks down diabetic mouse FMO3, inhibits the level of TMAO, and can improve insufficiency of insulin secretion.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of FMO3 in the preparation of drugs for promoting glucose-stimulated insulin secretion. Background technique [0002] Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia caused by insufficient insulin secretion and / or insulin resistance. Diabetes is a global health problem, and the number of cases is increasing year by year. The International Diabetes Federation predicts that by 2040, the number of people with diabetes will increase to 642 million. In addition, due to the high incidence of diabetes, the mortality rate caused by complications of diabetes is also increasing year by year. [0003] Most type 2 diabetes is secondary to obesity-related insulin resistance. Type 2 diabetes develops after years of prediabetes or impaired glucose tolerance. Type 2 diabetes manifests when insulin resistance is no longer compensated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12Q1/02A61K49/00
CPCC12Q1/26G01N33/507G01N33/5038A61K49/0008G01N2333/90251C12N2503/02G01N2500/10
Inventor 李平平孔丽娟赵其锦姜茜崔冰柳星峰马春晓侯少聪
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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