阅读说明：本技术 Gphb5糖蛋白激素在降脂和改善胰岛素敏感中的应用 (Application of GPHB5 glycoprotein hormone in reducing blood fat and improving insulin sensitivity ) 是由 赵子建 萧耿苗 李芳红 赵正刚 千爱君 张馨丹 王帅 于 2020-04-13 设计创作，主要内容包括：本发明提供了GPHB5糖蛋白激素在制备减少肥胖以及降脂药物,或治疗肥胖人群中葡萄糖不耐受、胰岛素抵抗或糖尿病的药物中的应用；应用包括利用从组织中纯化或重组表达并纯化的GPHB5蛋白,通过注射或口服,使肥胖人群减肥降脂,或者使肥胖人群改善胰岛素敏感,降低血糖；本发明依据于GPHB5糖蛋白激素在减肥减脂中的作用,揭示一种垂体调控体重和脂肪代谢的新机制。(The invention provides an application of GPHB5 glycoprotein hormone in preparing medicines for reducing obesity and reducing blood fat, or medicines for treating glucose intolerance, insulin resistance or diabetes in obese people; the application comprises that GPHB5 protein purified or recombined and expressed from tissues and purified is used for reducing weight and fat of obese people or improving insulin sensitivity and reducing blood sugar of the obese people through injection or oral administration; the invention discloses a novel mechanism for regulating body weight and fat metabolism by pituitary based on the action of GPHB5 glycoprotein hormone in weight reduction and fat reduction.)

Use of GPHB5 glycoprotein hormone in the manufacture of a medicament for reducing obesity and lowering lipid levels.

2. The use of claim 1, wherein the GPHB5 glycoprotein hormone causes weight loss.

3. The use of claim 1, wherein the GPHB5 glycoprotein hormone reduces the total body fat content without a significant change in non-fat content.

Use of GPHB5 glycoprotein hormone in the manufacture of a medicament for the treatment of glucose intolerance, insulin resistance or diabetes in obese people.

5. The use of any one of claims 1-4, wherein the GPHB5 glycoprotein hormone is a GPHB5 glycoprotein hormone purified from tissue or expressed recombinantly.

6. The use according to any one of claims 1 to 5, wherein the GPHB5 glycoprotein hormone is formulated for oral or injectable administration.

7. A fat lipid-lowering drug comprising a GPHB5 glycoprotein hormone, or a vector or cell expressing a GPHB5 glycoprotein hormone.

8. The medicament of claim 7, wherein the pharmacological effect comprises a decrease in body weight or a decrease in systemic fat content without a significant change in fat content.

9. A medicament for treating glucose intolerance or insulin resistance in obese humans comprising a GPHB5 glycoprotein hormone, or a vector or cell expressing a GPHB5 glycoprotein hormone.

10. The medicament according to any one of claims 7 to 9, which is in an oral or injectable dosage form.

Technical Field

The invention relates to the field of metabolic system diseases, and particularly provides application of GPHB5 glycoprotein hormone in preparing medicines for reducing obesity and reducing lipid or treating glucose intolerance or insulin resistance of obese people; the invention provides a glycoprotein hormone GPHB5 with weight-losing and fat-reducing effects, and discloses a novel mechanism for regulating weight and fat metabolism by pituitary.

Background

Obesity is a very common cause of death prevention and is one of the most important public health problems in the 21 st century. At present, the prevalence rate of obesity of adults and children is increasing, and women are more frequent than men. From 1979 to 2016, obese adult women increased from six thousand to nine million to three hundred million, and obese men increased from three thousand to two hundred and eight thousand to one million. Obesity can bring a series of complications, such as type 2 diabetes, cardiovascular diseases, infertility, asthma, sleep apnea syndrome and the like, is also related to the occurrence of malignant tumors such as breast cancer, rectal cancer, prostate cancer and the like, and seriously affects the health and the life quality of Chinese people.

GPHB5 is a subgroup of the group members of the glycoprotein hormone family, CGH, secreted mainly by the corticotropin-secreting cells of the anterior pituitary. Glycoprotein hormone family members include Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Thyroid Stimulating Hormone (TSH), Chorionic Gonadotropin (CG), and CGH, a family member of glycoprotein hormones that was identified as novel in 2002. In mammals, glycoprotein hormones play a key role in gonadal and thyroid function, gonadotropins regulate growth and differentiation of the ovaries and testes, and TSH is essential for energy balance in the body.

Studies have shown that GPHB5 is capable of binding to the TSH receptor (TSHR) and that GPHB5 and GPHA2, after binding to form a heterodimeric CGH, activate TSHR to produce cAMP 4-fold more strongly than TSH. In addition, activation of adipose tissue TSHR promotes hydrolysis of fatty triglycerides, which in turn reduces fat content and weight loss. Therefore, GPHB5 is likely to be combined with adipose tissue TSHR, stimulates triglyceride hydrolysis, and can be used as a potential molecular target for losing weight and reducing fat.

Disclosure of Invention

The invention mainly solves the problem of providing a new mechanism for losing weight and reducing fat and treating glucose intolerance or insulin resistance in obese people; on the basis of the existing research, the invention provides the application of GPHB5 glycoprotein hormone in obesity lipid-lowering and treatment of glucose intolerance or insulin resistance in obese people; based on the role of the glycoprotein hormone GPHB5 in weight loss and fat loss, a novel mechanism for the pituitary to regulate body weight and fat metabolism is disclosed.

The invention relates to a glycoprotein hormone glycoprotin hormonebeta 5(GPHB5) with the efficacy of reducing weight and fat.

In one aspect, the invention provides the use of a GPHB5 glycoprotein hormone in the manufacture of a medicament for reducing obesity and lowering lipid levels.

Further, the GPHB5 glycoprotein results in weight loss.

Further, the GPHB5 glycoprotein reduced the systemic fat content, but did not significantly change the non-fat content.

In another aspect, the invention provides the use of a GPHB5 glycoprotein hormone in the manufacture of a medicament for the treatment of glucose intolerance, insulin resistance or diabetes in obese humans.

The GPHB5 glycoprotein hormone can be GPHB5 glycoprotein hormone purified in tissues or expressed by recombination.

The GPHB5 glycoprotein hormone can be prepared into oral or injection dosage forms.

In another aspect, the invention provides an obese lipid-lowering medicament comprising GPHB5 glycoprotein hormone, or a vector or cell expressing GPHB5 glycoprotein hormone.

Further, the drug effect includes a decrease in body weight, or a decrease in systemic fat content without a significant change in fat content.

In another aspect, the invention provides a medicament for treating glucose intolerance or insulin resistance in obese humans, comprising a GPHB5 glycoprotein hormone, or a vector or cell expressing a GPHB5 glycoprotein hormone.

The medicine can be in oral or injection dosage forms.

The GPHB5 glycoprotein hormones of the present invention include GPHB5 glycoprotein hormones of various biological origins.

The GPHB5 glycoprotein hormone of the present invention may be GPHB5 glycoprotein hormone purified from tissues or expressed recombinantly using various known isolation and purification methods.

The GPHB5 glycoprotein hormone of the present invention does not exclude the presence of other amino acid sequences, as long as it does not prevent it from functioning as a GPHB5 glycoprotein hormone.

The pharmaceutical composition of the present invention may contain various excipients, vectors, expression cells, which are known or unknown in the pharmaceutical/biological field, and those skilled in the pharmaceutical/biological field can design corresponding dosage forms according to the prior knowledge.

Other drugs that lower blood lipid, treat glucose intolerance or insulin resistance are not excluded from the applications and drugs in the art, and one skilled in the art can try to combine these components with the GPHB5 glycoprotein hormone and verify its effect.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a novel mechanism for regulating body weight and fat metabolism by pituitary based on the action of GPHB5 glycoprotein hormone in weight reduction and fat reduction. The invention is helpful for losing weight, enhancing insulin sensitivity and improving blood fat metabolism by injecting or GPHB5 patients with low recombinant hormone level.

Drawings

FIG. 1 shows that GPHB5 knockout verification shows that GPHB5mRNA expression levels in testis, brain and heart of a wild type mouse and a knockout mouse are detected by qPCR, GPHB5mRNA in three tissues of the knockout mouse is obviously lower than that of the wild type mouse, and the knockout mouse basically does not express. n.d. represents not detectable.

FIG. 2 shows the weight gain curves of GPHB5 knock-out mice (-/-) and littermate wild-type mice (WT).

FIG. 3 is a comparison of the body types of GPHB5 knock-out mice (-/-) and littermate wild-type mice (WT).

FIG. 4 is a comparison of total body Fat content (Fat Mass) and non-Fat content (Lean Mass) for GPHB5 knockdown mice (-/-) and littermate wild type mice (WT).

FIG. 5 is a comparison of the glucose tolerance (GTT) of GPHB5 knockdown mice (-/-) and littermate wild type mice (WT).

FIG. 6 is a comparison of GPHB5 knock-out mice (-/-) and littermate wild type mice (WT) insulin tolerance (ITT).

FIG. 7 is a schematic representation of the expression vector pBudCE4.1-GPHB5/GPHA2 plasmid.

FIG. 8 is a schematic diagram of the expression vector pcDNA3.1-GPHB5-L-GPHA2 plasmid.

FIG. 9 shows the Western Blot detection of recombinant GPHB5/GPHA2 protein.

FIG. 10 shows the change of cAMP in cells after 3T3-L1 cells were stimulated by recombinant protein by ELISA.

In the figure, p < 0.05 represents significant difference; p < 0.01, the difference is very significant; p < 0.001, the difference was extremely significant; the differences are very significant, representing p < 0.0001.

Detailed Description

In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

In the examples, loss of GPHB5 glycoprotein hormone resulted in weight gain, specifically, loss of GPHB5 resulted in an increase in systemic fat content, while loss of GPHB5 resulted in glucose intolerance and insulin resistance.

The application comprises the following steps: the GPHB5 protein which is purified from tissues or is expressed and purified by recombination is used for leading obese patients to lose weight and reduce fat by injection or oral administration; or the GPHB5 protein which is purified from tissues or is expressed and purified by recombination is used for improving the insulin sensitivity and reducing the blood sugar of obese patients by injection or oral administration.

Example 1 construction, validation and correlation index analysis of GPHB5 knock-out mice

The Genbank number of the mouse GPHB5 gene is NM-175644.3, the Genbank number of the human GPHB5 gene is NM-145171.4, and the total length of the GPHB5 gene is about 3900 bp. The GPHB5 knockout mouse is entrusted to Seisaku Biotechnology Limited company, the exons 2 and 3 of the GPHB5 gene are knocked out by a CRISPR/Cas9 gene knockout technology, and 3340bp bases are knocked out to obtain 2 GPHB5 knockout mice which are marked as line1 and line 2. The first 10 sites which are possible to miss the target are detected by DNA sequencing, and the result shows that neither line1 nor line2 miss the target, which indicates that the GPHB5 knockout mouse is successfully constructed.

Through the heterozygote and heterozygote breeding, GPHB5 knock-out mice and littermate wild-type mice are obtained. The mouse and the testis, brain and heart of the wild type mouse are knocked out by GPHB5, the mRNA level of GPHB5 is detected, as shown in figure 1, the result shows that the GPHB5 knocked-out mouse does not express GPHB5mRNA basically.

Body weight comparison of GPHB5 knockdown mice to littermate wild-type mice:

feeding mice in SPF barrier environment, placing 3-4 male mice in the same cage, and feeding with the same feed and sterile water. Body weights were recorded once a week starting after mice matured for 6 weeks, as shown in fig. 2 and 3. After the mice age 18 weeks, the weight of the GPHB5 knockout mice is obviously higher than that of littermate wild-type mice, and the weight of the GPHB5 knockout mice is 6g more than that of the wild-type mice at 31 weeks, which indicates that the GPHB5 deletion can cause obesity.

Analysis of systemic fat content and non-fat content:

the body composition analyzer Echo MRI of the Huijia biological products Co., Ltd is adopted to detect the fat content and the non-fat content of the whole body of the GPHB5 knock-out mouse and the littermate wild-type mouse. As shown in fig. 4, the body fat content of the GPHB5 knockout mouse is significantly higher than that of the littermate wild-type mouse, while the non-fat content is substantially consistent, indicating that the increase in body weight of the GPHB5 knockout mouse is mainly due to fat accumulation.

Sugar tolerance analysis:

GPHB5 knock-out mice and littermate wild-type mice were fasted for 12 hours and fasting blood glucose values were measured with a roche glucometer. According to 2g/kg body weight, 20% glucose is injected intraperitoneally, blood glucose values of mice 15min, 30min, 60min, 90min, 120min and 150min after injection are measured by a Roche glucometer, and as shown in figure 5, the result shows that the glucose tolerance of the mice knocked out by GPHB5 is obviously lower than that of littermate wild type mice.

Insulin tolerance assay:

after the GPHB5 knock-out mouse and the littermate wild-type mouse are fasted for 6 hours, fasting plasma glucose is measured by a roche glucometer, 0.8IU/kg body weight of the mouse is intraperitoneally injected with 0.2IU/ml of insulin diluent, blood glucose values of the mouse are measured by the roche glucometer 15min, 30min, 45min, 60min, 75min and 90min after injection, and the blood glucose value measured before injection is 100% as an insulin tolerance curve, as shown in fig. 6. The results indicate that GPHB5 knockdown mice are not insulin sensitive.

Example 2 expression and functional analysis of recombinant GPHB5/GPHA2 protein in mammalian cells construction of expression vector pBudCE4.1-GPHB5/GPHA2 plasmid:

the complete gene sequences of GPHB5 and GPHA2 were synthesized by Nanjing Kinsley and codon humanization optimized. The gene of interest was cloned into the pBudCE4.1 vector, with the GPHB5 gene cloned between Hind III/BamH I sites of the vector and the GPHA2 gene cloned between Not I/Xho I sites of the vector, as shown in FIG. 7. The pBudCE4.1-GPHB5/GPHA2 plasmid is transformed into MJ109 Escherichia coli, and a single clone is picked for plasmid enzyme digestion identification and DNA sequencing method to verify the correctness of the inserted sequence. The pBudCE4.1 plasmid is a commonly used vector for expressing double genes in mammalian cells, the vector carries a His label, a V5 label and a myc label sequence, and shows according to a gene insertion site that after recombinant protein is expressed, the GPHB5 protein carries myc and His labels, and the GPHA2 protein carries V5 and His labels, so that the vector can be used for purification and detection.

Construction of expression vector pcDNA3.1-GPHB5-L-GPHA2 plasmid:

a fusion gene containing the signal peptide of GPHB5, the mature peptide fragment, the mature peptide of linker peptide L, GPHA2 and the His tag was synthesized and cloned between EcoR I/Not I sites of the pCDNA3.1 vector as shown in FIG. 8. Among them, the protein sequences of the linker peptide L are two: a conventional linker peptide GGGGSGGGGSGGGGS, and a CTP peptide segment PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ capable of extending the half-life of the recombinant protein.

Expression and functional analysis of recombinant GPHB5/GPHA2 protein in mammalian cells:

the constructed expression vector pBudCE4.1-GPHB5/GPHA2 and expression vector pcDNA3.1-GPHB5-L-GPHA2 were transfected into mammalian cells including HEK 293T cells, 293F cells, CHO cells. FIG. 9 shows the detection results of Western Blot experiment on the culture medium supernatant after 293T cells transfected by expression vector pBudCE4.1-GPHB5/GPHA2 were cultured for 2 days, indicating that the expression of GPHB5/GPHA2 recombinant protein was successful. The culture medium containing the recombinant protein is concentrated by 5 times through an ultrafiltration tube, the culture medium is purified by a His tag purification kit and is used for stimulating 3T3-L1 cells for 20min, 60min and 90min, and the cAMP level in the cells is detected by ELISA (enzyme-Linked immuno sorbent assay), so that compared with a control group, the cAMP is increased after the expressed GPHB5/GPHA2 recombinant protein stimulates for 20min and 60min, and as shown in figure 10, the expressed recombinant protein is proved to have activity.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.