CN108671223B

CN108671223B - Application of FHL3 in preparing medicament for treating insulin resistance

Info

Publication number: CN108671223B
Application number: CN201810596619.1A
Authority: CN
Inventors: 张云霞; 刘晓萌; 黄丽; 于月; 王慧慧
Original assignee: Zhoukou Normal University
Current assignee: Zhoukou Normal University
Priority date: 2018-06-11
Filing date: 2018-06-11
Publication date: 2021-08-20
Anticipated expiration: 2038-06-11
Also published as: CN108671223A

Abstract

The invention relates to application of FHL3 in preparing a medicament for treating insulin resistance. Specifically, the invention relates to an application of FHL3 in preparing a medicament for regulating the expression of a glucose transporter GLUT4 in cells, and also relates to an application of a cell strain for stably expressing the FHL3 gene in preparing a medicament for treating insulin resistance. By over-expressing FHL3 at the cellular level, it was found that FHL3 inhibits uptake and utilization of glucose by mature myotubes and that the amount of glucose transporter expression is significantly reduced. FHL3 transgenic mice gained weight, insulin sensitivity decreased significantly, and simultaneously glucose tolerance was impaired. The functional research of the FHL3 has great application prospect and higher reference value in the development of the medicine for improving and treating the insulin resistance.

Description

Application of FHL3 in preparing medicament for treating insulin resistance

Technical Field

The invention relates to application of FHL3 in preparing a medicament for treating insulin resistance.

Background

Skeletal muscle is the primary site of glucose utilization and storage and plays an important role in the balance of carbohydrate metabolism. Skeletal muscle insulin resistance is one of the pathogenesis factors of type 2 diabetes. There are several important protein factors in skeletal muscle that can regulate insulin resistance in skeletal muscle. For example, muscle secreted Irisin (Irisin) is activated byUCP1(unsaturated protein 1) Gene expression improves insulin sensitivity and glucose metabolism (Bortm P et al, A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and hermogenesis. Nature2012, 481:463-468. doi: 10.1038/nature 10777); PGC-1 alpha (Peroxisome proliferator-activator-gamma coactivator) is regulated and controlledGLUT4Gene expression is involved in the glucose metabolism (Michael LF, Wu Z, Cheatham RB, et a1. Restoration of an insulin-sensitive glucose transporter (GLUT 4) gene expression in muscle cells by the transcriptional coactivator PGC-1. Proc Natl Acad Sci USA2001, 98: 3820-3825); hypoxia inducible factor HIF-1 (Hypoxia inductive factor-1) has important regulatory roles in Skeletal Muscle Glucose Metabolism and Insulin sensitivity (G targets SW et al, Hypoxia in Combination With Muscle context Insulin Action and Glucose Metabolism in Human Skelet membrane via the HIF-1. alpha. pathway. Diabetes, 2017, 66: 2800-2807. doi: 10.2337/db 16-1488). Therefore, the discovery of new drug targets for improving skeletal muscle insulin resistance opens up more ways for preventing and treating type 2 diabetes.

The FHL (four and a half LIM domain) protein family is a protein of LIM-only subfamily in the LIM protein family, and consists of four half LIM structural domains, and LIM cannot directly interact with DNA and can only regulate gene expression through interaction with other proteins or transcription factors. The family of proteinsFHL1AndFHL3genes are highly expressed in skeletal muscle (Zuo B et al, Full-length cDNA, expression pattern and association analysis of the gene FHL3 gene.Asian-Aust J Anim Sci2007, 20: 1473-; the previous research of the applicant of the present project shows that: FHL3 regulates the conversion between myofiber types through the interaction of MyoD and CREB (Cyclic-AMP responsive element binding protein 1) proteins (Zhang YX et al, FHL3 differential definitions of MyHC isoforms through MyoD and pCREB.Cellular Signalling, 2016, 28: 60-73). While different muscle fiber types differ in insulin sensitivity and glucose uptake capacity, type I fiber (expression) MyHC 1/slowGene) High ratio of soleus muscle to insulin (Pataky MW et al, High-Fat D)iet-Induced Insulin Resistance in Single Skeletal Muscle Fibers is Fiber Type Selective. Sci Rep, 2017, 7: 13642.). Therefore, FHL3 may play a role in insulin sensitivity and sugar metabolism, and although a plurality of interacting proteins of FHL3 are found and preliminary study on the possible physiological action is carried out, more biological functions of FHL3 are still to be studied and found deeply. However, no studies have been reported to date on the role of FHL3 in the development of glucose metabolism and insulin resistance. The invention further researches the biological function of FHL3, thereby establishing the relationship between FHL3 and glucose metabolism and insulin sensitivity and providing a new target for improving insulin resistance.

Disclosure of Invention

The invention aims to provide application of FHL3 in preparing a medicament for treating insulin resistance, namely application of FHL3 in preparing a medicament for treating insulin resistance, or application of FHL3 in treating insulin resistance by methods such as gene therapy and the like. The invention relates to a new medical application of FHL 3.

The inventors have unexpectedly discovered in their studies that FHL3 can be modulatedGLUT4The expression of the gene further influences the sensitivity of the individual to insulin and the uptake and utilization degree of glucose, and provides a brand new scheme for the clinical treatment of insulin resistance. Based on this finding, the present inventors completed the invention.

In one aspect, the invention provides FHL3 in preparation for use in modulationGLUT4The use of a gene for the production of a medicament for expression and activity in mammalian cells.

Based on the use of the first aspect of the invention, the second aspect of the invention provides the use of FHL3 in the manufacture of a medicament for the promotion of glucose absorption and utilisation.

In a third aspect, the invention provides an expression vector comprising a nucleotide sequence encoding FHL3 (seq id No. 1). In one embodiment, the vector is a plasmid. In another embodiment, the virus is a lentivirus.

In a fourth aspect, the invention provides the use of an expression vector according to the third aspect of the invention in the preparation of a medicament for modulating a glucose transporterGLUT4Gene in breast feedUse in a medicament for expression in a milk animal cell.

In a fifth aspect, the invention provides the use of an expression vector according to the third aspect of the invention in the manufacture of a medicament for inhibiting insulin resistance in a mammal.

A sixth aspect of the invention providesFHL3The C2C12 mouse myoblast cell line with gene stably expressed is applied in preparing medicine for treating insulin resistance.

The invention will be further described in the next step.

The first aspect of the invention providesFHL3In preparation for conditioningGLUT4The use of a gene for the preparation of a medicament for expression in a mammalian cell.

The FHL3 is a known protein, which is called as follows: four and a half LIM domains 3, which can be amplified by PCR to obtain the FHL3 CDS sequence encoding the entire coding region of 289 amino acids (SEQ ID NO: 2).

Glucose metabolism depends on Glucose uptake by cells, and Glucose uptake by cells can be realized by virtue of Glucose Transporter (Glucose Transporter 4) GLUT4 transport function on cell membranes. The low expression level or activity of GLUT4 leads to the decrease of glucose uptake by skeletal muscle cells, and is an important molecular phenotype of insulin resistance.

Through intensive research, the FHL3 is found to inhibit the mammalian cellsGLUT4Gene expression, the mammalian cells can be any tissue and organ cells that use glucose as an energy source. In another aspect, the mammalian cell is both in vivo and ex vivo.

Based on the use of the first aspect of the invention, the second aspect of the invention provides the use of FHL3 in the manufacture of a medicament for promoting glucose uptake and utilisation.

Insulin Resistance (IR) refers to a decrease in the sensitivity of the target organs (skeletal muscle, fat, liver) for Insulin action to Insulin action, and is a causative factor in a variety of metabolic diseases, resulting in decreased uptake and utilization of glucose by cells.

The "mammalian cell" described in the second aspect may be either in a somatic cell or an ex vivo cell; "mammal" refers to a subject suffering from insulin resistance as described herein. Including but not limited to swine, dogs, cats, cattle, sheep, and other domestic animals and humans.

In a third aspect, the invention provides an expression vector comprising a nucleotide sequence encoding FHL 3. Such vectors include, but are not limited to, any plasmids and viruses that can be used as expression vectors. The virus may be a virus known in the art to be useful as a vector, the virus being a lentivirus

In a fourth aspect, the invention provides the use of an expression vector according to the third aspect of the invention in the preparation of a medicament for modulatingGLUT4Use of a gene in a medicament for expression in a mammalian cell. The expression vector may be a component of the medicament.

In a fifth aspect, the invention provides the use of an expression vector of the third aspect of the invention in the manufacture of a medicament for inhibiting insulin resistance in a mammal, said expression vector being a component of said medicament.

According to the present invention, a medicament containing FHL3, or an expression vector containing a nucleotide sequence encoding FHL3, can be administered to a subject in need of treatment by any route known in the art, including, but not limited to: systemic administration, topical administration to the skin, topical administration to the focal area, and the like. In particular, the routes of administration include, but are not limited to: intravenous, intraarterial, intramuscular, oral, intraperitoneal, pulmonary inhalation or insufflation, parenteral, transmucosal, nasal, rectal.

According to the present invention, the drug may be in a form of a drug for administration well known in the art, including pharmaceutical compositions, pharmaceutical preparations, and the like. Specifically, the pharmaceutical composition is, for example, a preparation for parenteral administration, such as a preparation for local injection and a preparation for systemic injection, and the specific dosage form may be, for example, a solution for injection, a powder for injection, or the like. For the practice of the present invention, the medicament of the present invention is preferably a formulation for parenteral administration, including, but not limited to, topical injection formulations and systemic injection formulations, and specific dosage forms include, but are not limited to, injectable solutions and injectable powder injections.

According to the results of the present study, it has been found that FHL3 gene expression vectors obtained from vectors, particularly lentiviruses, are capable of inhibiting glucose uptake in cells and of regulating the activity of the cellsGLUT4The expression of the gene thus affects insulin sensitivity.

The invention discovers that FHL3 participates in the regulation of the generation of insulin resistance and the imbalance of glucose homeostasis for the first time. Therefore, FHL3 can provide a new drug target for clinical treatment and improvement of insulin resistance.

Drawings

FIG. 1 is a nucleotide sequence expression vector encoding FHL 3; A) the vector is an empty vector pCDH-CMV-MCS-EF 1-copGFP; b) Is a recombinant vector pCDH-CMV-MCS-EF1-copGFP-FHL 3.

FIG. 2 shows the result of enzyme digestion identification of FHL3 recombinant lentiviral vector; m represents marker;

lanes

1, 2, 3, 4 represent recombinant plasmids; lane 5 shows empty vector.

FIG. 3 shows fluorescence microscopy results (10) 48h after fluid exchange of 293FT transfected cells: A) the method comprises the following steps Transfecting 293FT cells with pCDH-CMV-MCS-EF1-copGFP lentivirus; B) the method comprises the following steps pCDH-CMV-MCS-EF1-copGFP-FHL3 lentivirus transfected 293FT cells.

FIG. 4 shows the GFP fluorescence intensity (. times.4) measured by lentivirus titer.

FIG. 5 shows the lentivirus infection of C2C12 cellsFHL3The overexpression effect of (3).

FIG. 6 is overexpressionFHL3Post-genic C2C12 cell changes in glucose uptake.

FIG. 7 is overexpressionFHL3GeneGLUT4 The amount of mRNA expressed varies.

FIG. 8 shows the weight change of FHL3 transgenic mice.

FIG. 9 is a fasting blood glucose and random blood glucose test for mice; A) fasting blood glucose; B) random blood glucose; C) a mouse glucose tolerance test; D) insulin tolerance test.

Detailed Description

The present invention will be further described with reference to the following examples. The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; general equipment, materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 construction and characterization of FHL3 recombinant Lentiviral vectors

After total RNA of the mouse myoblast C2C12 is extracted by a Trizol method, cDNA is obtained by reverse transcription. Primers were designed based on the mouse mRNA Sequence (NCBI Reference Sequence: NM-010213.3) as follows: f: CGGAATTCGTCATGAGCGAGGCATTTGAC (SEQ ID NO: 3) and R: CGGAATTCTTATCAGGGGCCTGCTTGGCTG (SEQ ID NO: 4). The CDS sequence of the mouse FHL3 gene is amplified by a PCR method by using the primers and taking the mouse cDNA as a template, and the PCR reaction system is shown in a table 1:

table 1 PCR reaction system.

The PCR reaction conditions are shown in Table 2:

PCR reaction conditions

After the PCR product is purified and cloned, sequence identification is carried out, and sequencing work is finished by Shanghai Bioengineering technology service company Limited. The NCBI blast sequence alignment shows 100% similarity. The recombinant plasmid is cut and identified by restriction enzymes BamHI and EcoRI, and detected by 1.5% agarose gel electrophoresis, and if the cut product of the recombinant plasmid contains a fragment less than 1000bp (870bp) as shown in FIG. 1 pCDH-CMV-MCS-EF1-copGFP-FHL3FHL3Gene CDS sequence, large fragment is linear vector; the pCDH-CMV-MCS-EF1-copGFP empty vector has only a linear vector fragment (FIGS. 1, 2).

Example 2 Lentiviral packaging, concentration and Titer assay

293FT cells are cultured and inoculated on a 10 cm dish, the recombinant plasmid, a packaging plasmid psPAX2 and an envelope plasmid pMD2.g transfect the 293FT cells together by using a Neofect transfection reagent, the liquid is changed after 6 h, and the large-scale green fluorescent protein expression of the cells is observed under a fluorescence microscope 48h and 72 h after the liquid is changed. Almost all cells were green, and the transfection efficiency was approximately 95% or more by observing green fluorescence (FIG. 3). After 72 h, the cells were visibly rounded and ruptured. Centrifuging at 3000g for 10 min, and collecting virus supernatant for 48h and 72 h.

Concentrating the virus by using a Filter device, adding 12 ml of virus liquid into the Filter device each time, centrifuging for 30min at 4000g, wherein the concentration liquid in the Filter device after centrifugation is about 150 mu l, concentrating all virus liquid after centrifuging for several times, and storing the concentrated virus liquid serving as seed virus in a refrigerator at minus 80 ℃ for later use. The invention adopts a fluorescence method to determine the virus titer, the day before the determination, 293FT cells required by the determination of the titer are inoculated in a 24-well plate, the cell fusion degree is about 80 percent, and each well is about 5 multiplied by 10⁵And (4) cells. Diluting the seed virus by 100 times with complete culture medium, infecting 293FT cells, changing the culture medium after 2 h, and observing the fluorescent expression condition after 2 days. FHL3 recombinant lentivirus and control lentivirus titers were approximately 10 based on GFP expression (FIG. 4)⁸TU/ml, cells can be subsequently infected.

Viral titer (TU/mL) = number of fluorescent cells ÷ total cells ÷ 5 × 1.5 × 10⁵×10³。

Example 3 Stable expressionFHL3Screening and identification of Gene C2C12 cell line

1X 10 per empty C2C12 cells⁴Cells were seeded into 96-well plates. After 12 hours of culture, the control lentiviral fluid was diluted with a medium (DMEM +10% FBS +8 ug/ml polybrene) at MOI =20, 50, 100, 200, 300, 400, and the diluted lentiviral fluid was added into each well of the cell, and the fluorescence intensity of GFP was observed after culturing in an incubator for 72 hours. Finally, the optimal MOI value of the C2C12 mouse myoblast lentivirus is determined to be 200.

MOI value = viral titer (TU/mL) x viral volume (mL)/cell number

C2C12 cells were seeded in 24-well plates for Puro working concentration screening, and Puro was diluted with medium at concentrations of 0 ug/ml, 2 ug/ml, 5 ug/ml, 8 ug/ml, 10 ug/ml. The final minimum lethal concentration was 2 ug/ml.

One day before virus infection, C2C12 cells are inoculated in a 24-well plate, the cells are fused to 60% -80%, culture medium (DMEM +10% FBS +8 ug/ml polybrene) is used for diluting virus liquid of an experimental group and a control group according to MOI =200, the virus liquid infects C2C12 cells, GFP expression is observed after 48 hours, and Puro antibiotic with the concentration of 2 ug/ml is used for screeningFHL3Cell lines with stably expressed genes were screened for 10 days, and the medium containing 2 ug/ml Puro was changed every 3 days. And (4) continuously subculturing the screened and surviving cells, and freezing and storing. Extracting total RNA from a part of cells by a Trizol method, and detecting the total RNA by a qPCR (quantitative polymerase chain reaction) technologyFHL3The expression level of gene mRNA.FHL3The expression quantity of gene mRNA is up-regulated, indicating that the gene is over-expressedFHL3The gene was successful (FIG. 5), and the selected C2C12 mouse myoblasts wereFHL3The cell strain with stably expressed gene can be used for subsequent gene function research.

Example 4 FHL3 inhibits the uptake of glucose into C2C12 mature myotubes

Example 3 aboveFHL3C2C12 cells and control cells with genes stably expressed are inoculated into a 12-well plate, and after 6 days of DMEM +2% horse serum induction differentiation of mature myotubes, 1 mu M Dexamethasone (DEX) is respectively used for intervention for 24 h (DEX can induce C2C12 cells to generate insulin resistance phenomenon). The experimental settings were experimental group (PCDH-FHL 3) + Dex, control group (PCDH) + Dex, experimental group (PCDH-FHL 3) + absolute ethanol (AA), control group (PCDH) + absolute ethanol (AA) (DEX was dissolved in absolute ethanol). After that, the Insulin stimulates the mature myotube for 30 minutes, and the glucose uptake of the myotube is detected by a glucose oxidase method. The results show that the method has the advantages of high yield,FHL3the glucose uptake in mature myotubes with gene overexpression was significantly reduced and there was no increase in glucose uptake by cells under insulin stimulation (figure 6), indicating that FHL3 decreased the sensitivity of cells to insulin.

Example 4 FHL3 inhibition of glucose transportersGLUT4Gene expression

Example 3 aboveFHL3C2C12 cells with gene stably expressed and control cells are inoculated into a 12-well plate and induced by horse serumCollecting cells after differentiation for 6 days, extracting total RNA of the cells by a Trizol method, and detecting overexpression by a qPCR methodFHL3Downstream gene of post-gene insulin signaling pathwayGLUT4The expression level. Glucose metabolism depends on glucose uptake by cells, which can be achieved by GLUT4 transport function on cell membranes (Mulder AH, Tack CJ, Olthaar AJ, Smits P, sweet FC, Bosch RR. Adrenergic receptor stimulation assays in-stimulated glucose uptake 3T3-L1 adipocyte by inhibiting GLUT4 transport).Am JPhysiol Endocrinol Metab 289: E627-E633, 2005.). A low level of expression or reduced activity of GLUT4, which results in decreased glucose uptake by skeletal muscle cells, is an important molecular phenotype of insulin resistance (Hidestsu Sakagami, Yuichi Makino, Katsutoshi Mizumoto, et al, Loss of HIF-1. alpha. improvirs GLUT4 translocation and glucose uptake by the skeletal muscle cells. Am J Physiol Endocrinol Metab2014, 306, E1065-E1076). The qPCR experiment result shows that the overexpression is compared with the control groupFHL3Post-genic, in C2C12 mature myotubesGLUT4The mRNA expression was significantly reduced (fig. 7). FHL3 may be inhibitedGLUT4Gene expression affects intracellular glucose transport, resulting in the development of insulin resistance.

Example 5 FHL3 transgenic mice develop an imbalance in insulin resistance and glucose homeostasis

Cell experiment results show that FHL3 causes the reduction of the insulin sensitivity of cells. The invention further studies the relationship between FHL3 and insulin sensitivity at the in vivo level by means of a mouse model. Wild type C57BL/6 mice andFHL3transgenic mice were 8 each, male in sex, fed on a normal diet, and their body weights were recorded weekly. Results of the experiment it was found that transgenic mice gained weight (fig. 8) during the experiment blood glucose measurements, fasting glucose tolerance (GTT) and Insulin Tolerance Test (ITT) were performed. Fasting and random blood glucose were significantly higher than wild type mice (fig. 9). The GTT and ITT experimental methods are as follows:

the glucose consumption of the mouse GTT experiment is generally 1.5 or 2g/kg, the glucose consumption of the invention is 2g/kg, and a 20% glucose solution is prepared by using physiological saline. The mice are changed into a clean cage for fasting at 5 pm before the experiment, the mice are fasted for 16 hours, the mice keep normal drinking water, the weight of each mouse is weighed up to 9 am of the next day, the fasting basal blood glucose is measured by a glucometer, and the measured value is determined as the blood glucose value of 0min (the operation is as gentle as possible, so that the mice are not frightened excessively); after the mice were acclimated for 30min, the mice were gently grabbed, and glucose solutions were injected into the mice according to standard intraperitoneal injection procedures with a 1ml syringe (the injection volume was determined according to the weight of the mice, and 0.01ml per g of body weight) at the moment of completion of the injection, timing was started, and the blood glucose values at various time points of each mouse were measured at 15min, 30min, 60min, 90min, and 120 min.

In the mouse ITT experiment, the dosage of insulin is generally 1U/kg, and the insulin is prepared by normal saline. Mice were replaced in clean cages at 9 am and fasted for 4 hours, and mice were kept drinking water normally until 1 pm, and their weights were weighed. Measuring blood sugar before insulin injection (same as GTT experiment), and allowing mice to adapt for 30min before insulin injection, and injecting insulin solution into abdominal cavity (same as GTT experiment); blood glucose values were measured at various time points in each mouse 15min, 30min, 45min, and 60min after injection. And finally analyzing the data. GTT and ITT experiments showed that FHL3 decreased insulin sensitivity and impaired glucose tolerance in mice (figure 8).

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Claims

1. Nucleotide sequence expression vector for coding FHL3 in preparation of glucose transporter for regulatingGLUT4The use of genes in medicaments for expression and activity in mammalian cells; the FHL3 is: NCBI FHL3 open reading frame nucleotide sequence NM — 010213.3 (mus).

2. The use according to claim 1, wherein the medicament is for inhibiting intracellular activity in a mammalian cellGLUT4Gene expression and activity control glucose uptake and utilization.

3. The use according to claim 1, wherein said mammalian cell is selected from the group consisting of a somatic cell and an ex vivo cell.

4. The application of the nucleotide sequence expression vector for coding FHL3 in preparing the medicine for improving and treating insulin resistance, FHL3 is used as a medicine target to regulate the uptake and utilization of glucose by inhibiting the activity of GLUT4, and the FHL3 is as follows: NCBI FHL3 open reading frame nucleotide sequence NM — 010213.3 (mus).