CN111920955A

CN111920955A - Application of Grb10 as key negative regulator of beta cell dysfunction

Info

Publication number: CN111920955A
Application number: CN202011025653.7A
Authority: CN
Inventors: 张晶晶
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2020-06-09
Filing date: 2020-09-25
Publication date: 2020-11-13
Also published as: CN111494635A

Abstract

The invention relates to the field of biological medicines, in particular to application of Grb10 as a key negative regulator of pancreatic islet beta cell dysfunction. The invention studies the physiological function of Grb10 in beta cells. The experimental results show that Grb10 is highly expressed in human and mouse pancreas and islets, and is highly expressed in islets of diabetic mouse model and in the islets of the elderly. In addition, β cell-specific knockout of Grb10 increased β cell mass and β cell function in High Fat Diet (HFD) mice by promoting β cell maturation and inhibiting β cell dedifferentiation. The invention highlights the important role of the interaction of Grb10-mTORC1 in regulating the dedifferentiation of beta cells and provides new insights for the regulation mechanism of the characteristics and functions of the beta cells.

Description

Application of Grb10 as key negative regulator of beta cell dysfunction

The present application claims priority from chinese patent application filed on 09/06/2020, having application number 202010517856.1 and entitled "use of Grb10 as a key negative regulator of beta cell dysfunction", the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of biological medicines, in particular to application of Grb10 as a key negative regulator of beta cell dysfunction.

Background

A reduction in the number of beta cells and/or an impaired function of beta cells is one of the mechanisms by which type 1 diabetes (T1D) and type 2 diabetes (T2D) occur. However, the underlying mechanism of diabetes, beta cell depletion, remains to be fully established.

Studies have shown that mature and differentiated beta cells are not in an immortalized state, which can change with environmental changes. There is increasing evidence that islet beta cell dedifferentiation plays a key role in the loss of diabetic beta cells. Dedifferentiated beta cells lose their properties and insulin secretion function, with a concomitant down-regulation of beta cell-specific transcription factors and up-regulation of endocrine precursor surface markers. The de-differentiation of beta cells into neuronal 3(Ngn3) -like progenitor cells and transdifferentiation into other endocrine cells is associated with the development of human T2D. However, the molecular mechanisms that regulate the characteristics of beta cells remain unknown. Studies have shown that glucose transporter 2(Glut2) and the pancreatic duodenal homeobox 1(PDX1) are required for maintaining beta cell properties. However, under normal and abnormal metabolic conditions, it is currently unclear whether other key molecules exist to regulate the identity of beta cells.

Growth factor receptor-bound protein 10(Grb 10) is a protein that negatively regulates insulin and mTORC1 signaling in insulin target cells (PH) and Src homology 2 (SH 2) domain proteins. Grb10 has been identified as a diabetes susceptibility gene, but its tissue-specific and mechanism of action in diabetes remains elusive. Our previous studies showed that pancreatic Grb10 knockout mice increase beta cell mass by promoting beta cell proliferation and protecting beta cells from streptozotocin-induced apoptosis. However, the exact mechanism by which Grb10 regulates islet beta cell homeostasis and function is not clear.

mTORC is a serine/threonine kinase that is critical to cell growth and organ development. Deletion of the beta cell-specific mTORC1 results in beta cell failure due to defects in cell proliferation, apoptosis, and insulin secretion. Consistent with these findings, intraperitoneal injection of rapamycin significantly reduced the number of β cells in mice. On the other hand, activation of the mTORC1 signaling pathway increases beta cell mass. Although these findings suggest that the mTORC1 signaling pathway plays a critical role in regulating beta cell mass, the specific mechanism by which mTORC1 signaling pathway regulates beta cell mass and function remains unclear.

Disclosure of Invention

In view of this, the present invention provides the use of Grb10 as a key negative regulator of beta cell dysfunction.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of Grb10 as a key negative regulator of beta cell dysfunction.

In some embodiments of the invention, the beta cell dysfunction comprises beta cell dedifferentiation, loss of beta cell properties, beta cell exhaustion.

The invention also provides application of Grb10 inhibition in preparation of a medicine for preventing and/or treating beta cell dysfunction.

The invention also provides application of inhibition of Grb10 in preparation of drugs for improving glucose tolerance of animals.

The invention also provides application of a blocking agent of Grb10 inhibition or Grb10 regulation signal pathway in preparation of a drug for improving beta cell function and preventing and/or treating diabetes.

In some particular embodiments of the invention, said inhibition of Grb10 comprises a function of reducing the expression of Grb10, knockout and/or down-regulation of the Grb10 gene.

In some embodiments of the invention, said inhibition of Grb10 promotes beta cell proliferation.

In some embodiments of the invention, said inhibition of Grb10 inhibits beta cell dedifferentiation.

In some embodiments of the invention, the signaling pathway regulated by Grb10 enhances beta cell mass and improves beta cell insulin secretion function.

In some embodiments of the invention, the signaling pathway regulated by Grb10 is the mTORC1 signaling pathway.

The invention studies the physiological function of Grb10 in beta cells. The experimental results show that Grb10 is highly expressed in human and mouse pancreas and islets, and is highly expressed in islets of diabetic mouse model and in the islets of the elderly. In addition, β cell-specific knockout of Grb10 increased β cell mass and β cell function in High Fat Diet (HFD) mice by promoting β cell maturation and inhibiting β cell dedifferentiation. Mechanistically, beta cell-specific gene knockout of Grb10 upregulates the mTORC1 signaling pathway, resulting in increased expression of transcription factors that promote beta cell maturation and decreased expression of transcription factors that promote endocrine pancreatic development. The invention highlights the important role of the interaction of Grb10-mTORC1 in regulating the dedifferentiation of beta cells and provides new insights for the regulation mechanism of the characteristics and functions of the beta cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the experimental results of Effect example 1; wherein, (A) qRT-PCR detects the gene expression level of Grb10 in human muscle, brain and pancreatic islets; (B) WB assay Grb10 protein expression levels in human brain, liver, muscle, fat, heart, kidney, spleen and pancreas; tublin is used as an internal reference; (C) human pancreatic tissue sections were examined for Grb10 and immunofluorescence of insulin; (D) WB was used to detect Grb10 protein expression levels in islets of C57BL/6J mice fed Normally (ND) and High Fat (HFD) for 16 weeks; (E) WB detecting the protein expression level of Grb10 in the islets of 3-month-old control mice and ob/ob mice; (F) WB detecting protein expression level of Grb10 in islets of male db/+ mice and diabetic db/db mice of 3 months old; D. the beta-Actin in the F picture is used as an internal reference; (G) detecting the gene expression levels of Grb10, Sox9, Ngn3 and PDX1 in MIN6 cells cultured in medium containing 5.5mM Normal Glucose (NG) or 40mM High Glucose (HG); (H) detecting Grb10 and Ngn3 protein levels in MIN6 cells cultured for 2 days under 5.5mM (NG) or 40mM (HG) glucose conditions; (I) protein expression levels of Grb10 in human islets younger than 18 years of age and human islets older than 18 years of age; beta-Actin is used as an internal reference; data are expressed as mean ± SEM; p < 0.05;

FIG. 2 shows the experimental results of Effect example 2; wherein (a) immunofluorescence detects fluorescence of 8-week old male loxp (wt) mice and Grb10KO (KO) mouse pancreas sections Grb10 (red) and insulin (as a beta cell marker) (green); marking and quantifying the pancreatic area of each slice by a brightness extraction method; (B, C) high-fat fed 16-week male control mice (WT) and Grb10KO (KO) mice tested for beta cell to islet ratio (B) and alpha cell to islet ratio (C); (D, E) high-fat feeding of 16-week male control mice (WT) and Grb10KO (KO) mice to measure the average beta cell mass (D) and the average alpha cell mass (E); (F) HFD was fed to 16 weeks of loxP control mice (WT; n-8), GTT of control mice (Ins 2-cre; n-6) and Grb10KO (KO; n-8) to measure glucose tolerance; blood glucose was measured from the tail vein at 0, 15, 30, 60 and 120 minutes after intraperitoneal injection of glucose (2g/kg body weight); p compared to loxP control group (WT).)<0.05，**P<0.01，***P<0.001; compared with the cre mouse, the mouse has the advantages that,^#P<0.05; (G) the ITT of control mice (Ins 2-cre; n-6) and Grb10KO (KO; n-8) from the control group, which were fed with HFD for 16 weeks (WT; n-8), examined insulin tolerance; (H) the hyperglycosylation clamp measures the glucose infusion rate of normally fed control and Grb10KO (KO) mice (n-5/group); (I) control mice and Grb10KO (KO) mice fed normally during the hyperglycosylation phase (n-5/group) were tested for serum insulin levels; experimental data represent mean ± SEM; p<0.05；**P<0.01；***P<0.001 (one-way repeated measures analysis of variance);

FIG. 3 shows the experimental results of Effect example 3; wherein, (A) qRT-PCR analyzes the gene expression of islets Grb10, Ngn3, Glut2, PDX1 and Ins1 of control group mice fed with high fat for 16 weeks and Grb10KO (KO) mice; (B) WB analysis protein expression levels of islets Glut2, PDX1, Ngn3 of control mice and Grb10KO (KO) mice fed with high fat for 16 weeks, with β -Actin as an internal control; (C) immunofluorescence assay control mice and Grb10KO (KO) mice insulin and Glut2 fluorescence of pancreatic sections under ND and HFD feeding conditions; (D) GFPMIN6 cells and Grb10 OE MIN6 cells constructed on MIN6 cells using adenovirus Ad-GFP (GFP) or Ad-Grb10(Grb10 OE) and detecting the gene expression levels of Glut2, PDX1, NKX6-1, Ins1 and Ins 2; (E) WB detected protein expression levels of Glut2 and PDX1 in GFP MIN6 cells and Grb10 OEMIN 6 cells; beta-Actin is used as an internal reference; the relative expression of proteins by Glut2 and PDX1 in control GFPMIN6 cells and Grb10 OE MIN6 cells is shown in the histogram; data represent mean ± SEM; p < 0.05; p < 0.01; p < 0.001;

FIG. 4 shows the experimental results of effect example 4; wherein, (A) qRT-PCR analysis of Grb10, Ngn3 and Sox9 gene expression in islets of Grb10KO (KO) mice and control mice fed with HFD for 16 weeks; (B) WB analysis of protein expression of Ngn3 and Sox9 in islets of control mice fed with HFD for 16 weeks and Grb10KO (KO) mice; beta-Actin is used as an internal reference; (C, D) after Grb10 is over-expressed in MIN6 cells, (C) the gene expression of Grb10, Ngn3, Sox9 in GFP MIN6 cells and Grb10 OEMIN 6 cells is detected by qRT-PCR; (D) WB detects the protein expression of Ngn3 and Sox9 in GFPMIN6 cells and Grb10 OEMIN 6 cells; beta-Actin is used as an internal reference; (E) immunofluorescence insulin and Sox9 fluorescence were detected from pancreas sections of control mice and Grb10KO (KO) mice under HFD feeding conditions, and data are presented as mean ± SEM; p < 0.05; p < 0.01; p < 0.001;

FIG. 5 shows the experimental results of effect example 5; wherein (A) after the control group mice Grb10KO (KO) fed by HFD respectively treat a control solvent and rapamycin, the WB is used for checking the protein expression of Grb10, Glut2, PDX1, Ngn3, S6, 4EBP1 and S6 phosphorylation at Ser235 site and 4EBP1 phosphorylation at Thr37/46 site in the islets of four groups of mice, and beta-Actin is used as an internal reference; (B, C) INS-1 cells were treated with siRNA (NC) and Grb10 specific siRNA (siGrb10), respectively, for 48 hours; (B) qRT-PCR detection of gene expression conditions of Grb10, Ngn3 and Sox9 is carried out 24 hours after the cells are infected with a control shRNA or sh-raptor lentivirus; (C) cells are treated by rapamycin for 24 hours and then are cracked, and WB detects the phosphorylation of Glut2, PDX1, S6 and S6 kinase at Thr389 site, 4EBP1 and 4EBP1 at Thr37/46 site and the expression of Grb10 protein; beta-Actin is used as an internal reference; (D) the glucose tolerance of mice is detected by using GTT after HFD-fed control group mice and Grb10KO (KO) mice respectively treat a control solvent and rapamycin; (WT; n-10), Grb10KO (KO; n-6), (WT + Rap; n-10) and (KO + Rap; n-7); (E) beta cell mass was measured in four groups of mice after HFD-fed control mice and Grb10KO mice treated with control solvent and rapamycin, respectively; (WT; n-7), Grb10KO (KO; n-6), (WT + Rap; n-8), and (KO + Rap; n-6); (D, E) data represent mean ± SEM; (D) p <0.05, P <0.01, P < 0.001; (F) immunofluorescence was measured for fluorescence expression of insulin and Glut2 in pancreas sections of four groups of mice treated with control solvent and rapamycin, respectively, in HFD-fed control mice and Grb10KO (KO) mice; (G) mode diagram: the mechanism by which Grb10 regulates beta cell dedifferentiation suggests: the long-term HFD feeding can induce the dedifferentiation of beta cells, is accompanied by the down-regulation of the expression of beta cell-specific genes and proteins and up-regulates the expression of genes and proteins related to secretion precursors, Grb10 negatively regulates mTORC1 signal pathways, and after the beta cell-specific knockout of Grb10, mTORC1 activates the signal pathways, inhibits the dedifferentiation of the beta cells and promotes the proliferation of the beta cells.

Detailed Description

The invention discloses application of Grb10 as a key negative regulator of beta cell dysfunction, and a person skilled in the art can appropriately modify process parameters for realization by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In this study, we explored the mechanism by which Grb10 regulates beta cell mass. We found that β cell Grb10 gene knock-out enhances the ability of pancreatic islet β cells to stimulate insulin secretion (GSIS) by glucose, which is associated with a sustained state of cell maturation under long-term HFD feeding conditions. Mechanistically, knockout of Grb10 results in activation of the mTORC1 signaling pathway, inhibition of beta cell dedifferentiation, maintenance of beta cell identity and increase of beta cell mass. Our studies indicate a key role of the Grb10-mTORC1 axis in regulating beta cell dedifferentiation and provide clues for the development of potential new targets for the effective treatment of beta cell failure, the major cause of diabetes.

Maintaining the mature nature of islet beta cells is critical to maintaining beta cell mass and glucose homeostasis in the body. Unlike other terminally differentiated cells, beta cells may lose their properties and dedifferentiate under certain pathophysiological conditions, leading to beta cell failure and consequent diabetes. However, the mechanisms that regulate the properties of beta cells remain to be further elucidated. In this study, we found that specific deletion of mouse Grb10 in beta cells inhibited HFD-induced expression of precursor genes such as Ngn3 and Sox9 (fig. 4). The lack of Grb10 in β cells also increased the expression of genes characteristic of the mature differentiation of β cells, such as insulin, PDX1, Glut2, etc. (fig. 3). These results indicate that inhibition of beta cell dedifferentiation contributes to an increase in beta cell mass and insulin content in HFD fed Grb10ko (ko) mice.

Several signaling pathways, such as the Hedgehog, Wnt, Notch and NF-. kappa.b signaling pathways, have been found to regulate beta cell dedifferentiation. We found that the beta cell-specific knockout of Grb10 had no significant effect on activation of Wnt, Notch and NF- κ B signaling pathways. On the other hand, Grb10 gene knockout greatly increased phosphorylation of islet beta cell S6 at Ser235 site and 4EBP1 at thr37/46 site, suggesting activation of mTORC1 signaling pathway. Inhibition of the mTORC1 signaling pathway by rapamycin or Raptor knockdown increased the expression of the endocrine precursor marker Ngn3 (fig. 5A-B). Consistent with this result, intraperitoneal injection of rapamycin also significantly reduced β -cell mass (fig. 5E) and glucose intolerance in Grb10ko (ko) mice. These results indicate that activation of the mTORC1 signaling pathway plays an important role in inhibiting the differentiation of beta cells to a precursor cell-like state.

Maintenance of beta cell mass is regulated by many complex processes, and insufficient beta cell mass can lead to type 1 diabetes and type 2 diabetes. While islet transplantation can complement beta cell mass, this approach is limited by the lack of donors. Therefore, the development of effective therapeutic methods for promoting beta cell proliferation is crucial for beta cell transplantation. Unfortunately, although human cells can be induced to proliferate in vitro, most proliferating beta cells lose the property of expressing and secreting insulin. Lineage tracking indicates that beta cells undergo rapid loss of beta cell phenotype when they promote human cell proliferation in vitro. Activation of the Hedgehog signaling pathway promotes proliferation of beta cells, but also results in de-differentiation of beta cells. The research shows that the knockout of the Grb10 gene in the beta cell can not only promote the proliferation of the beta cell, but also obviously inhibit the dedifferentiation of the beta cell, and the fact that the targeting Grb 10-regulated beta intracellular signal pathway is probably an effective strategy for enhancing the quality of the beta cell and improving the insulin secretion function of the beta cell is suggested.

Taken together, we determined that Grb10 is a key regulator of beta cell dedifferentiation. Furthermore, we found that inhibition of Grb10 expression in beta cells can inhibit the dedifferentiation of beta cells by activating mTORC1 signaling pathway, thereby maintaining beta cell characteristics and improving cell function. Increasing beta cell dedifferentiation and apoptosis and decreasing cell proliferation are key factors in the pathogenesis of T1D and T2D. Understanding the regulatory mechanisms of cell dedifferentiation and determining key regulatory factors in the dedifferentiation process of beta cells can provide valuable information for the formulation of new strategies for T1D and T2D treatment.

The gene name is: grb10

The species are as follows: mouse

ID：14783

Amino acid sequence: as shown in SEQ ID No. 1.

The gene name is: grb10

The species are as follows: human being

ID：2887

Amino acid sequence: as shown in SEQ ID No. 2.

The raw materials and reagents used in the application of the Grb10 provided by the invention as the key negative regulator of the beta cell dysfunction are all available in the market.

The invention is further illustrated by the following examples:

example 1 human study

Islets from 13 human organ donors were used in this study, including 6 minors (under 18 years) and 7 adults (over 18 years). Islets less than 18 years of age were compared to Grb10 expression from adult donors (older than 18 years of age). Human pancreas was obtained from dead non-diabetic donors by the urinary surgery organ/liver transplant department of the Hunan Yabi Hospital, southern China university.

Human islets were isolated by digestion with collagenase P, the adipose tissue surrounding the pancreas, lymph nodes, blood vessels and fascia were carefully cleaned and the human pancreas was trimmed, the pancreatic canula was inflated with a 5mL syringe, and Hanks Balanced Salt Solution (HBSS) containing 1mg/mL of collagenase P was injected through the pancreatic duct in an amount equivalent to twice the weight of the pancreas. The pancreas was removed and placed in collagenase P solution and digested at 37 ℃ for about 20-30 minutes, and the tubes were placed in an ice bath (4 ℃) to inactivate collagenase P and preserve the islets. The digestion was stopped by adding Hanks buffer, then the pancreas was washed 3 times with RPMI-1640 medium, human islet medium and dithizone dye were added and islets were selected under a microscope for isolation. The ethical committee of xiang yadi hospital, central university, approved the use of isolated human islets (protocol MSRC2016 LF).

For WB and qRT-PCR experiments, we used human brain, liver, muscle, fat, heart, kidney, spleen and pancreas from urological organ/liver transplantations in yaja, xiang, university, central southern china. The samples obtained were snap frozen and stored at-80 ℃ until use. The ethical committee of xiang yadi hospital, central university, approved the use of isolated human islets (protocol MSRC2016 LF).

Example 2 animal models

Beta cell specific Grb10 knockout mice Grb10KO (KO) were generated by breeding homozygous Grb10 female (loxP) mice on a C57BL/6J background with female homozygous Ins2-Cre mice on a C57BL/6J background. Ins2-Cre is expressed in pancreatic beta cells, but not in hypothalamic neurons. loxP littermates and Ins2-Cre littermates were used as controls (WT). The Ins2-Cre mice were provided by octopine (university of Tianjin medical science). All mice were housed in specific pathogen free facilities (SPF) at the animal care center of yaja, xiang, university, central south, with a 12 hour bright-dark cycle. All procedures for animal use were performed according to the Hunan Yay second Hospital animal Care and the Committee for animal Care and use in the Central and south university.

Example 3 body weight, food intake and body composition

Mice were fed either a plain diet (ND) containing 19% protein, 5% fat and 5% fiber (Hunan silake jingda laboratory animal Co Ltd) or a High Fat Drink (HFD) containing 20% protein; 60% fat; 20% carbohydrate (D12492, research Diets Inc.) for 16 weeks. Mice body weight and food intake were monitored weekly at the same time point. Mice were analyzed for lean mass and fat mass by MQ Minispec 7.5HZ live mouse Analyzer (MinispecLF 50; BRUKER Optik GmbH; Germany).

EXAMPLE 4 high sugar tongs

Awake, normally-fed 4-month-old loxP wild-type littermates (WT) and male Grb10KO (KO) mice were fasted overnight and anesthetized with ketamine (IP) (100mg/kg body weight) and xylazine (10mg/kg body weight) by intraperitoneal injection, 4-5 days prior to the clamp experiment, an indwelling catheter was inserted into the right internal jugular vein of the mice, the mice were housed in individual cages, and post-operative recovery and weight gain were monitored. After an overnight fast, a2 hour high glucose clamp experiment was performed in conscious mice with 10% glucose infusion at a variable rate to raise and maintain plasma glucose concentrations at 400mg/dL at 350-. Blood samples (20 μ L) were collected every 5 to 10 minutes to measure plasma glucose and insulin levels.

Example 5 mouse islet isolation and insulin particle count

Mouse islets were isolated by collagenase P digestion. An overnight fasted loxP (WT) control littermate and male Grb10KO (KO) mice were anesthetized by intraperitoneal injection of tribromoethanol (1mL/40g body weight), mice were pancreas-distended with 3mL of a 1mg/mL solution of collagenase P (Sigma Chemical of St. Louis, Mo.; collagenase P was added to Hank's buffered saline (HBSS)), the pancreas was removed and digested at 37 ℃ for about 12 minutes, then 30mL of HBSS was added to stop digestion, and finally washed 3 times with 20mL of RPMI-1640 medium, islet medium and dithizone dye were added and islets were isolated under a microscope.

For insulin particle counting, islets were isolated and fixed using 1% glutaraldehyde, embedded in epon, and tissue blocks (0.1-1.5 mm)³) In the presence of 1% OsO₄0.1mol/L sodium cocoate buffer (pH 7.4) for 1 hour at room temperature, washed in a cocoate buffer, then washed in distilled water, and then subjected to a 2% uranyl acetate double distilled water treatment for 1 hour. The sections were dehydrated in gradient alcohol and then placed in 100% propylene oxide. The blocks were incubated overnight in propylene oxide-propylene oxide (1: 1) at room temperature, then changed to 100% ethylene oxide and polymerized overnight. Sections (90nm) were cut on a Reichert-Jung UltracutE microtome, collected on a slotted grid, and stained with 2% uranyl acetate and lead citrate. Pictures were taken by Jeol1230 transmission electron microscope (Jeol, peabody, massachusetts). Three different empty particles (EG), immature particles (IG) and mature secretory particles (MSG) were counted accordingly.

Example 6 islet proliferation assay and TUNEL assay

Mouse pancreatic sections were analyzed by double staining with anti-ki 67 (Abcam; 16667) and anti-insulin (Sigma; I2018) antibodies. Insulin positive beta cells were counted and the percentage of total insulin positive nuclei detected for loxp (wt) control mice and Grb10KO (KO) mice ki67 and insulin double positive nuclei. The percentage of ki67 and the ratio of insulin double positive nuclei to total insulin positive nuclei were determined as the proliferation rate of loxp (wt) control mice and Grb10KO (KO) mice. The total ki67 positive cell number was quantified by using an anaerotactic method to ensure that the same ki67 labeled cells were not counted twice on adjacent sections and that the pancreatic section area remained consistent for each animal.

Male loxp (wt) control and Grb10KO (KO) mouse pancreas sections fed with HFD for 16 weeks were labeled with in situ cell death detection kit and insulin antibody and HFD-induced apoptosis was determined by immunofluorescence using an olympus inverted microscope (IX71) and captured with a Sport II digital camera. Six mice per group were used for (TUNEL) analysis (Beyotime C1090). More than 2500 insulin positive cells were counted per mouse. Apoptosis was calculated from TUNEL and insulin double positive nuclei to total insulin positive nuclei.

EXAMPLE 7 administration of rapamycin

Male loxP (WT) control and Grb10KO (KO) mice were fed HFD for 16 weeks every 3 days with an intraperitoneal injection of 5mg/kg rapamycin or control group solvent for 12 days. Rapamycin was dissolved in 100% ethanol at a concentration of 5mg/mL, diluted to 0.5mg/mL in a carrier solution of 5% Tween-80 and 5% PEG-400 in PBS, and then used after filtration.

Example 8 RNA interference in INS-1 cells

Cells were cultured in antibiotic-free medium for 24 hours and then transfected with siRNA targeting Grb10 (Ribobio; siG161129043512) or scrambled siRNA (Ribobio) at a final concentration of 25nM for 24 hours. Lipofectamine was used according to the manufacturer's instructions^TM3000 (ThermoFisher; L3000015) for transfection assay. After overnight transfection, the medium was replaced with conventional medium.

Example 9 Generation of adenovirus and Adenoviral infection

Adenovirus encoding GFP and Grb10 adenovirus were generated by using pAdEasy system. MIN6 cells infected adenovirus at a multiplicity of infection of 30. The efficiency of adenovirus infection was assessed by fluorescent GFP expression using a fluorescence microscope 24 hours post infection.

Example 10 real-time PCR

Total RNA was isolated from cells using Trizol (life technology) according to the manufacturer's instructions. The RNA concentration was determined using a UV-Vis spectrophotometer Q5000. Quantitative PCR reactions were performed using SYBR mixtures (Roche) and quantified using an applied biosystems 7900HT sequence detection system. Duplicate runs of each sample were performed and β -Actin was used as an internal reference to determine relative expression levels.

Example 11 GTT, ITT, Immunofluorescence (IF).

The Glucose Tolerance Test (GTT) was performed by injecting glucose (2g/kg body weight i.p.) into overnight fasted mice. Blood was withdrawn from the

tail vein

0, 15, 30, 60 and 120 minutes after the glucose injection, and the blood glucose level of the mice was measured with a glucometer (one touch; Bionime Corp.).

Insulin resistance test (ITT) was performed by injection of human insulin (0.75U/kg body weight i.p.) into 4 hour fasted mice. At 0, 15, 30, 60 and 90 minutes after insulin injection, tail vein bleeds were initiated and serum insulin levels were determined using an insulin hypersensitivity enzyme immunoassay (Alpco Diagnostics, Slemm, NH).

Immunofluorescence (IF) experiments were performed by taking the pancreas of loxp (wt) control mice and Grb10KO (KO) mice, fixing overnight (DING GUO) with 4% paraformaldehyde, then dehydrating with 30% sucrose, embedding with o.c.t., and cutting 8 μm sections of pancreas using frozen sections for use. The sections were fixed, blocked, permeabilized and stained with specific antibodies-Grb 10 (homemade), 1: 50; insulin (Sigma I2018)1: 500; glut2(Millipore company 07-1402-I)1: 1000; ki67(abcam 16667)1: 500; sox9(Milipore AB5535)1:500, cell nuclei were identified using DAPI staining. The LSM 780 laser scanning confocal microscope multi-tracking setup detects the fluorescence signal using the corresponding fluorescent secondary antibody. Each channel uses the same pinhole diameter.

Example 12 statistics

The summary data represent mean ± SEM. Statistical analysis was performed using GraphPadPrism 7(GraphPad software). Statistical analysis of the data was performed by using unpaired t-test or ANOVA (single repeated measures ANOVA or two-way ANOVA with Bonferroni test after the fact). Statistical significance was set at P values P <0.05, P <0.01 and P < 0.001. P values less than 0.05 are considered statistically significant.

Effect example 1 whether expression of Grb10 in beta cells is regulated by metabolic stress

Grb10 was highly expressed in human islets (FIGS. 1A-C) and mouse islets, and in islets of diet-induced obese mice (FIG. 1D), ob/ob mice (FIG. 1E), and db/db mice (FIG. 1F). mRNA (fig. 1G) and protein (fig. 1H) levels of Grb10 were also increased in high concentration glucose-treated MIN6 cells. Considering that aging is a key risk factor for the prevalence of type 2 diabetes, we also examined the expression of Grb10 in islets isolated from non-adults and elderly. The elderly islet Grb10 protein expression was increased compared to minors (fig. 1I). The above data indicate that the expression of Grb10 is dynamically regulated by a variety of metabolic stimuli.

TABLE 1 FIG. 1D data

TABLE 2 FIG. 1E data

TABLE 3 FIG. 1F data

TABLE 4 FIG. 1G data

TABLE 5 FIG. 1H data

TABLE 6 FIG. 1I data

Effect example 2 knockout of the beta cell-specific Gene Grb10 improves the ability of the mouse beta cell GSIS and HFD-induced glucose intolerance

To determine the functional role of Grb10 in beta cells, we crossed female Grb10 flox (loxp) mice with male transgenic mice expressing Cre recombinase (Ins2-Cre) to generate beta cell-specific Grb10 knockout mice (Grb10 KO). By immunoblotting and immunofluorescence staining, we found that Grb10ko (ko) mice had a significant reduction in Grb10 protein levels in islets, while wild-type control mice had no significant reduction in Grb10 protein levels (fig. 2A and 4B). The Ins2-cre mediated deletion of Grb10 had no effect on Grb10 levels in other tissues including the hypothalamus. The weight, food intake, body composition and tissue weight of the Grb10KO (KO) mice were not significantly different from those of the control group. On the other hand, the β cell to islet ratio (fig. 2B) and the α cell to islet ratio (fig. 2C) of the Grb10ko (ko) mice were significantly increased and decreased, respectively. Consistent with these findings, Grb10ko (ko) mice showed more beta cell mass than control mice (fig. 2D). In contrast, the number of alpha cells was significantly reduced in Grb10KO (KO) mice (fig. 2E). Immunofluorescence experiments with the ki67 antibody showed that high fat diet Grb10ko (ko) mice proliferated significantly increased numbers of islet cells compared to control mice. The TUNEL method shows that Grb10 gene knockout reduces beta cell apoptosis. On the other hand, the over-expression of Grb10 increased the expression of clear-caspase-3, and inhibited the expression of cyclin D2. Suggesting that Grb10 overexpression may reduce the mass of beta cells by promoting apoptosis and inhibiting cell proliferation. The β -cell specific Grb10 knockout had no significant effect on insulin sensitivity (fig. 2G), but significantly increased glucose tolerance (fig. 2F), indicating that Grb10ko (ko) mice have improved glucose tolerance. High glucose clamp experiments showed that Glucose Infusion Rate (GIR) was higher in Grb10ko (ko) mice than in control mice (fig. 2H). Furthermore, the insulin secretion rate of the Grb10KO (KO) mice was higher than that of the control group mice (FIG. 2I). Transmission electron microscope observation results show that the number of insulin particles, especially the number of mature secretory particles in Grb10KO (KO) mouse cells is obviously increased. Taken together, these data indicate that Grb10 knockout cells can improve beta cell function.

TABLE 7 FIG. 2B data

Table 8 fig. 2C data

TABLE 9 FIG. 2D data

TABLE 10 FIG. 2E data

TABLE 11 FIG. 2F data

TABLE 12 FIG. 2G data

TABLE 13 FIG. 2H data

TABLE 14 FIG. 2I data

Effect example 3 inhibition of Grb10 expression in beta cells protects against HFD-induced loss of beta cell properties

Dedifferentiation causes the beta cells to lose their identity and return to a precursor state, resulting in a reduction in cell number and impaired insulin secretion. To determine the mechanism by which Grb10 regulates beta cell mass, we wanted to figure out whether Grb10 gene knock-out helps maintain beta cell properties. One of the features of beta cell function is the expression of Glut2(Slc2a2), PDX1, NK6 homeobox 1(Nkx6-1), and insulin 1/2. We found that HFD fed Grb10ko (ko) mice islet Glut2 and PDX1 mRNA levels were significantly higher than control mice (fig. 3A). The expression of the insulin genes Ins1 and Ins2 was also higher in the islets of the Grb10KO mouse (fig. 3A). Consistent with the above results, protein levels of Grb10ko (ko) islets Glut2 and PDX1 were significantly higher in mice than in control mice (fig. 3B). There was no significant difference between the control group fed normally and the Glut2 immunoreactivity of islets of Grb10KO (KO) mice, but insulin expression of islets of Grb10KO (KO) mice was significantly higher than that of WT mice (FIG. 3C). The expression of Glut2 and insulin was significantly reduced in WT mice islets 16 weeks after high fat feeding (fig. 3C). Interestingly, HFD-fed Grb10ko (ko) mice expressed higher Glut2 and insulin immunoreactivity, similar to ND-fed WT mice (fig. 3C). In contrast, adenovirus-mediated overexpression of Grb10 in MIN6 cells (Grb10 OE MIN6 cells) significantly reduced mRNA expression of Glut2, PDX1, Nkx6-1, Ins1, and Ins2 (fig. 3D). MIN6 cell Glut2 and PDX1 protein levels of Grb10 OE were significantly reduced (fig. 3E). These findings indicate that down-regulation of Grb10 promotes the ability to maintain beta cell characteristics.

Table 15 fig. 3A data

TABLE 16 FIG. 3B data

TABLE 17 FIG. 3D data

Table 18 fig. 3E data

Effect example 4 Grb10 cell-specific knockdown prevented HFD-induced beta cells from returning to the precursor state

To further confirm the potential role of Grb10 in beta cell dedifferentiation, we examined the expression of islet endocrine precursor cell markers in Grb10KO mice and normal control mice. The expression levels of endocrine islet progenitor cell markers Ngn3 and Sox9 in HFD-fed Grb10KO mouse islets were significantly lower than in the control group (fig. 4A). Protein levels of Ngn3 and Sox9 were also significantly reduced (fig. 3B and 4B). In contrast, MIN6 cells from Grb10 OE showed a significant increase in gene expression (fig. 4C) and protein expression (fig. 4D) of Ngn3 and Sox 9. Immunofluorescence analysis confirmed that insulin and Sox9 double positive cells were detected in the pancreas of control mice compared to Grb10ko (ko) mouse tissue (fig. 4E, arrow labeled). The above results indicate that Grb10 induces beta cell dedifferentiation by upregulating cellular endocrine precursor markers.

Table 19 fig. 4A data

Table 20 fig. 4C data

Table 21 fig. 4D data

Effect example 5 potential role of Signal pathway in beta cell dedifferentiation

Knockout of Grb10 in beta cells maintained maturation of beta cells in a mTORC1 dependent manner. A number of signaling pathways such as Hedgehog, Wnt, Notch, and NF-. kappa.b have been identified as signaling pathways that regulate cell dedifferentiation. To elucidate whether these signaling pathways are involved in Grb 10-induced cell dedifferentiation, we examined the expression of key components of these signaling pathways in Grb10 OE MIN6 cells and GFPMIN6 cells. Grb10 overexpression had no significant effect on the expression levels of key components in Notch1, Hes1, p-p65, p65 and the beta-catenin signaling pathway.

Based on the study of Grb10 negatively regulating mTORC1 and the insulin signaling pathway in mammalian cells, we examined the potential role of these signaling pathways in beta cell dedifferentiation. Glut2 and PDX1 gene expression were not altered following treatment of MIN6 cells with PI3 kinase inhibitor Ly 294002. Suggesting that the effect of Grb10 on beta cell dedifferentiation is independent of the insulin signaling pathway. On the other hand, we found that phosphorylation of the islets of Langerhans of Grb10KO (KO) mice at Ser235 site of S6 and at thr37/46 site of 4EBP1 was significantly enhanced upon glucose stimulation compared to WT mice. In addition, overexpression of Grb10 in MIN6 cells inhibited phosphorylation of glucose-stimulated S6 at the Ser235 site and 4EBP1 at the thr37/46 site. These results confirm the inhibitory effect of Grb10 on mTORC1 signaling pathway in cells. The increased Glut2 and PDX1 expression induced by the Grb10 knockout was inhibited by rapamycin treatment, indicating that mTORC1 signaling pathway has a promoting effect in modulating the β cell characteristics of Grb10ko (ko) mice (fig. 5A). In addition, siRNA inhibited Raptor from increasing the expression of endocrine precursor genes such as MIN6 cells Ngn3 and Sox9 (fig. 5B). Similarly, INS-1 cells inhibited increased expression of PDX1 and Glut2 following Grb10, while expression of PDX1 and Glut2 was decreased following rapamycin treatment (FIG. 5C). To further demonstrate the role of mTORC1 signaling pathway in modulating beta cell characteristics in vivo, we administered HFD to greb 10ko (ko) mice and control mice intraperitoneally with rapamycin. Rapamycin injection inhibited mTORC1 signaling pathway for 12 days, with significantly reduced phosphorylation of S6 at Ser235 site and 4EBP1 at thr37/46 site (fig. 5A). Rapamycin had no significant effect on insulin resistance and weight gain in mice, but the improvement in glucose tolerance was significantly reversed in Grb10ko (ko) mice compared to the control group (fig. 5D). The β -cell mass of the Grb10ko (ko) mice was significantly higher than the control mice, but the β -cell mass was significantly reduced after rapamycin use (fig. 5E). The Grb10 knockout caused an increase in Glut2 immunoreactivity, while rapamycin treatment significantly reduced Glut2 immunoreactivity (fig. 5F). The above data indicate that knockout of Grb10 promotes maintenance of beta cell properties by activating mTORC1 signaling pathway.

TABLE 22 FIG. 5B data

Table 23 fig. 5D data

Table 24 fig. 5E data

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Xiangya II Hospital of Zhongnan university

Application of <120> Grb10 as key negative regulator of beta cell dysfunction

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Met Ala Leu Ala Gly Cys Pro Asp Ser Phe Leu His His Pro Tyr Tyr

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Gln Asp Lys Val Glu Gln Thr Pro Arg Ser Gln Gln Asp Pro Ala Gly

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Pro Gly Leu Pro Ala Gln Ser Asp Arg Leu Ala Asn His Gln Glu Asp

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Asp Val Asp Leu Glu Ala Leu Val Asn Asp Met Asn Ala Ser Leu Glu

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Ser Leu Tyr Ser Ala Cys Ser Met Gln Ser Asp Thr Val Pro Leu Leu

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Gln Asn Gly Gln His Ala Arg Ser Gln Pro Arg Ala Ser Gly Pro Pro

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Arg Ser Ile Gln Pro Gln Val Ser Pro Arg Gln Arg Val Gln Arg Ser

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Gln Pro Val His Ile Leu Ala Val Arg Arg Leu Gln Glu Glu Asp Gln

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Gln Phe Arg Thr Ser Ser Leu Pro Ala Ile Pro Asn Pro Phe Pro Glu

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Leu Cys Gly Pro Gly Ser Pro Pro Val Leu Thr Pro Gly Ser Leu Pro

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Pro Ser Gln Ala Ala Ala Lys Gln Asp Val Lys Val Phe Ser Glu Asp

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Gly Thr Ser Lys Val Val Glu Ile Leu Ala Asp Met Thr Ala Arg Asp

180 185 190

Leu Cys Gln Leu Leu Val Tyr Lys Ser His Cys Val Asp Asp Asn Ser

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Trp Thr Leu Val Glu His His Pro His Leu Gly Leu Glu Arg Cys Leu

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Glu Asp His Glu Leu Val Val Gln Val Glu Ser Thr Met Ala Ser Glu

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Ser Lys Phe Leu Phe Arg Lys Asn Tyr Ala Lys Tyr Glu Phe Phe Lys

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Asn Pro Met Asn Phe Phe Pro Glu Gln Met Val Thr Trp Cys Gln Gln

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Ser Asn Gly Ser Gln Thr Gln Leu Leu Gln Asn Phe Leu Asn Ser Ser

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Ser Cys Pro Glu Ile Gln Gly Phe Leu His Val Lys Glu Leu Gly Lys

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Lys Ser Trp Lys Lys Leu Tyr Val Cys Leu Arg Arg Ser Gly Leu Tyr

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Cys Ser Thr Lys Gly Thr Ser Lys Glu Pro Arg His Leu Gln Leu Leu

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Ala Asp Leu Glu Asp Ser Asn Ile Phe Ser Leu Ile Ala Gly Arg Lys

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Gln Tyr Asn Ala Pro Thr Asp His Gly Leu Cys Ile Lys Pro Asn Lys

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Val Arg Asn Glu Thr Lys Glu Leu Arg Leu Leu Cys Ala Glu Asp Glu

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Gln Thr Arg Thr Cys Trp Met Thr Ala Phe Arg Leu Leu Lys Tyr Gly

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Met Leu Leu Tyr Gln Asn Tyr Arg Ile Pro Gln Gln Arg Lys Ala Leu

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Leu Ser Pro Phe Ser Thr Pro Val Arg Ser Val Ser Glu Asn Ser Leu

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Val Ala Met Asp Phe Ser Gly Gln Thr Gly Arg Val Ile Glu Asn Pro

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Ala Glu Ala Gln Ser Ala Ala Leu Glu Glu Gly His Ala Trp Arg Lys

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Arg Ser Thr Arg Met Asn Ile Leu Gly Ser Gln Ser Pro Leu His Pro

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Ser Thr Leu Ser Thr Val Ile His Arg Thr Gln His Trp Phe His Gly

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Arg Ile Ser Arg Glu Glu Ser His Arg Ile Ile Lys Gln Gln Gly Leu

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Val Asp Gly Leu Phe Leu Leu Arg Asp Ser Gln Ser Asn Pro Lys Ala

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Phe Val Leu Thr Leu Cys His His Gln Lys Ile Lys Asn Phe Gln Ile

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Leu Pro Cys Glu Asp Asp Gly Gln Thr Phe Phe Ser Leu Asp Asp Gly

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Asn Thr Lys Phe Ser Asp Leu Ile Gln Leu Val Asp Phe Tyr Gln Leu

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Asn Lys Gly Val Leu Pro Cys Lys Leu Lys His His Cys Ile Arg Val

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Ala Leu

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