CN107011427B

CN107011427B - Polypeptide for regulating energy metabolism and application thereof

Info

Publication number: CN107011427B
Application number: CN201710156032.4A
Authority: CN
Inventors: 任培根; 张键; 滕斌; 李健; 姚振宇
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Zhongke Xinjin Biotechnology Co Ltd
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2020-06-12
Anticipated expiration: 2037-03-16
Also published as: CN107011427A

Abstract

The present invention relates to polypeptides which modulate energy metabolism and their use in the manufacture of a medicament for the treatment of diseases associated with abnormal energy metabolism, particularly fat metabolism. The polypeptide of the present invention can reduce fat absorption, reduce fat accumulation in the liver and regulate fat metabolism, and has the advantage of being orally administrable as compared with general polypeptide products, and thus, can also be used as an active ingredient of health care products for regulating fat metabolism.

Description

Polypeptide for regulating energy metabolism and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a polypeptide for regulating energy metabolism and application thereof in preparing a medicament for treating diseases related to energy metabolism abnormality, particularly fat metabolism abnormality.

Background

Obesity is a major and growing health problem worldwide. Obesity is also a risk factor that induces the development of many common diseases such as atherosclerosis, hypertension, type II diabetes, dyslipidemia, coronary heart disease, osteoarthritis and various malignancies. It also causes more serious problems by reducing exercise capacity and quality of life. The occurrence of obesity and diseases caused by obesity are increasing in all developed countries.

Fatty liver refers to a lesion caused by excessive accumulation of fat in liver cells. The causes of fatty liver are many, such as alcoholism, improper diet, and long-term sitting. The incidence of the Chinese traditional medicine is increasing day by day, and the Chinese traditional medicine becomes a great threat to the health of people.

However, the current drugs and methods for treating obesity and diseases caused by obesity, including non-alcoholic fatty liver disease, have certain disadvantages, and thus there is still a need for the development of more effective drugs and methods with low side effects for the treatment of obesity and non-alcoholic fatty liver disease.

Osteocalcin is a vitamin K-dependent calcium-binding protein synthesized and secreted by osteoblasts, a non-collagen acidic glycoprotein, in which the vitamin K-dependent glutamic acid residues in the molecule are osteocalcin and Ca²⁺Important functional groups for binding^[1,2]。

Disclosure of Invention

The present inventors have surprisingly found a polypeptide derived from osteocalcin, which is capable of modulating energy metabolism; it has effects in reducing fat absorption, reducing liver fat, reducing blood lipid in peripheral blood, and reducing volume of fat cells, can effectively remove fat accumulated in liver cells, reduce blood lipid level, and reduce in vivo adipose tissue, and can be used for treating steatohepatitis, regulating hypertension and other cardiovascular diseases.

Some aspects of the invention relate to polypeptides that modulate energy metabolism and are represented by formula M₁-Z_a-M₂Is shown, in which:

M₁、M₂each independently is a polypeptide segment having no more than 5, 4, 3, 2, or 1 amino acid residue or is absent;

Z_ais Tyr-Leu-X₁-X₂-X₃-X₄-Gly-Ala-X₅-X₆-Pro-X₇-Pro-Asp-X₈-Leu-Glu-Pro, wherein:

X₁is Tyr, Asn, Asp or is absent,

X₂gln, Asn, His, Pro, Ser or absent,

X₃is Trp, Gly or absent,

X₄is Leu or is absent,

X₅is a Pro or a Ser,

X₆is Ala or Val, or a pharmaceutically acceptable salt thereof,

X₇is Tyr or Ser, and the amino acid is,

X₈is a Thr or a Pro group,

and said Z is_aOptionally with amino acid substitutions, insertions or deletions and the total number of amino acid substitutions, insertions and deletions does not exceed 4, preferably does not exceed 3, more preferably does not exceed 2, most preferably does not exceed 1.

Further, in some embodiments of the invention, Z is in the polypeptide_aSelected from one of the following:

SEQ ID NO.1：YLGASVPSPDPLEP，

SEQ ID NO.2：YLYQWLGAPVPYPDPLEP

SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，

SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，

SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，

SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，

SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，

SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，

SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，

SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。

further, in other embodiments of the present invention, M is present in the polypeptide₁、M₂Are not present; or in yet other embodiments, Z is in the polypeptide_aIs YLYQWLGAPVPYPDPLEP, M₁Is absent and M₂Arg, namely the amino acid sequence of the polypeptide is shown as SEQ ID NO. 3; in addition toIn some embodiments, Z is in the polypeptide_aIs YLGASVPSPDPLEP, M₁Is absent and M₂Is Thr, namely the amino acid sequence of the polypeptide is shown as SEQ ID NO. 18.

Further aspects of the invention also relate to polypeptides for modulating energy metabolism comprising at least 6 contiguous amino acids selected from any one of the following sequences and having a total number of amino acid residues of no more than 18, such as no more than 17, 16, 15, 14:

SEQ ID NO.1：YLGASVPSPDPLEP，

SEQ ID NO.2：YLYQWLGAPVPYPDPLEP

SEQ ID NO.3：YLYQWLGAPVPYPDPLEPR，

SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，

SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，

SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，

SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，

SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，

SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，

SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，

SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。

further, it comprises any of the following:

SEQ ID NO.12：PVPYPDPLEP，

SEQ ID NO.13：PYPDPLEP，

SEQ ID NO.14：PDPLEP，

SEQ ID NO.15：SVPSPDPLEP，

SEQ ID NO.16：PSPDPLEP。

further, it is

SEQ ID NO.12：PVPYPDPLEP，

SEQ ID NO.13：PYPDPLEP，

SEQ ID NO.14：PDPLEP，

SEQ ID NO.15：SVPSPDPLEP，

SEQ ID NO.16：PSPDPLEP。

Some aspects of the invention relate to pharmaceutically acceptable salts of the polypeptides of the invention.

Some aspects of the present invention relate to a polypeptide of the present invention or a pharmaceutically acceptable salt thereof having the effects of reducing fat absorption, lowering blood lipid levels, alleviating non-alcoholic fatty liver disease, and reducing adipose tissue in the body.

Some aspects of the invention relate to polynucleotides encoding the polypeptides described herein above.

Some aspects of the invention relate to a vector comprising the aforementioned polynucleotide.

Some aspects of the invention relate to host cells transfected with the aforementioned vectors and capable of producing the polypeptides of the invention under conditions in which the proteins can be expressed.

Some aspects of the invention relate to pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, as described above.

The polypeptide or its pharmaceutically acceptable salt or pharmaceutical composition described in the present invention has an effect of regulating energy metabolism, particularly fat metabolism.

Some aspects of the invention relate to the use of a polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disease associated with an abnormal energy metabolism, more preferably fat metabolism, which disease may benefit from decreased fat absorption, decreased blood lipids, increased fat consumption or decreased fat accumulation, such as obesity, type II diabetes, non-alcoholic fatty liver disease, insulin resistance, hypertriglyceridemia, hyperglycemia, hypercholesterolemia, atherosclerosis, coronary heart disease.

Some aspects of the present invention relate to methods of treating a disease associated with abnormal fat metabolism comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the present invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, which disease may benefit from decreased fat absorption, decreased blood lipids, increased fat consumption or decreased fat accumulation, for example, obesity, type II diabetes, non-alcoholic fatty liver disease, insulin resistance, hypertriglyceridemia, hyperglycemia, hypercholesterolemia, atherosclerosis, coronary heart disease.

In some embodiments, the polypeptide of the invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof may be administered with any of the pharmaceutical and method compositions known to be useful for treating diseases associated with abnormal fat metabolism.

In some embodiments, the polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, is used for the treatment of a disease associated with abnormal fat metabolism, which disease may benefit from decreased fat absorption, decreased blood lipids, increased fat consumption or decreased fat accumulation, such as obesity, type II diabetes, non-alcoholic fatty liver, insulin resistance, hypertriglyceridemia, high cholesterol, atherosclerosis, coronary heart disease.

Some aspects of the invention relate to the use of a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a health product for reducing body weight.

Some aspects of the present invention relate to a health product for reducing body weight comprising a polypeptide of the present invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the polypeptide of the present invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof may be administered by various conventional means, preferably orally.

The polypeptide can directly influence the absorption of fat, reduces inconvenience of conventional polypeptide administration by oral administration, has few residues of the polypeptide, greatly reduces the cost, and has very remarkable potential advantage as a medicament.

Brief Description of Drawings

FIG. 1 shows body weight comparison of Obesity and non-alcoholic fatty liver disease (DIO-NAFLD) models obtained 12 weeks after feeding C57BL/6 mice with high fat Diet to normal controls; normal Diet (ND) and High Fat Diet (HFD).

FIG. 2 shows DIO-NAFLD mice on a high fat diet injected with different concentrations of ISAP for 6 consecutive weeks₁Injection of ISAP after OCN (mouse osteocalcin) compared to control group (high fat diet only mice)₁Effect on mice. (A) ND control group, HFD control group and daily intraperitoneal injection of OCN or ISAP₁Changes in epididymal fat pads in the mice of the group; (B) average mass of epididymal fat pads in each group; (C) each group ofHematoxylin eosin (H) of epididymal fat pad tissue section&E) Dyeing; (D) according to H under double blind conditions&E stained sections the average adipocyte surface area obtained was calculated. Statistical analysis: and comparing with an HFD control group. P<0.05,**:P<0.01,***:P<0.001。

FIG. 3 shows DIO-NAFLD mice injected with different concentrations of ISAP for 6 consecutive weeks on a high fat diet₁After OCN, i.p. injection of ISAP compared to control group (high fat diet only mice)₁Effects on mouse liver. (A) Photographs of representative images of the appearance of the livers of each group of mice; (B) liver tissue section H of each group of mice&E, dyeing; (C) surface area of accumulated non-alcoholic fatty liver cells obtained under double-blind conditions after liver oil red O staining in each group of mice. Statistical analysis: for comparison with the HFD control group. P<0.05,**:P<0.01,***:P<0.001. Each group had 6 mice for analysis.

FIG. 4 shows DIO-NAFLD mice on a high fat diet injected with different concentrations of ISAP for 6 consecutive weeks₁After OCN, ISAP was injected compared to the control group (high fat diet only mice)₁The influence on the liver function and the blood fat of the mice. (A) Alanine Aminotransferase (ALT); (B) alkaline phosphatase (ALP); (C) aspartate Aminotransferase (AST); (D) total serum cholesterol (TC); (E) low Density Lipoprotein (LDL); (F) high Density Lipoprotein (HDL). Statistical analysis: comparison with HFD control group. P<0.05,**:P<0.01,***:P<0.001. #: compared with ND control group. P #<0.05,##:P<0.01,###P<0.001. Each group had 6 mice for analysis.

FIG. 5 shows intragastric administration of ISAP₁And the effect of OCN on the absorption of small intestine fat. After the wild type C57BL/6 mouse fed by ND is subjected to stomach filling and sterilization of olive oil (30 minutes), ISAP is injected into the abdominal cavity₁Euthanizing the mice 30 minutes after OCN and dissecting to obtain small intestine, cryosectioning jejunum proximal duodenum segment and staining with oil red O, hematoxylin counterstaining (a); the oil red O positive area was quantified by ImageJ software under double blind conditions and the ratio of this area to the total area of small intestine villi was calculated (B). Statistical analysis: compared to saline + olive oil. P<0.001.N＝3。

FIG. 6 shows an ISAP₁And ISAP₂Binding to human GPRC6A and receptor internalization, respectively. (A) Human GPRC6A was overexpressed in Hela cells; (B) ISAP₁OCN and OCN-22; and ISAP₂hOCN and hOCN-22 with the cell membrane of HeLa cells over-expressed by huGPRC 6A; (C) OCN labeled with Cy-5 (Cy5-OCN), OCN-22 labeled with Cy-5 (Cy5-Ocn22), ISAP labeled with Cy-5₂(Cy5-ISAP₂) Effect on GPRC6A internalization in Hela cells overexpressing GPRC 6A.

FIG. 7 shows administration of OCN, ISAP in transgastric gavage₁、ISAP₂、ISAP₃Effects on mice within the last seven weeks: (A) change in body weight of the mouse; (B) and comparing the content of triglyceride in the mouse excrement.

Figure 8 shows a comparison of the sequences of ISAPs from different species.

FIG. 9 shows intragastric administration of ISAP₄、ISAP₅、ISAP₆The effect on the absorption of fat in the small intestine. 15 ND-fed wild-type C57/b mice are divided into 5 groups of 3 mice each and three experimental groups, and the ISAP is respectively administered by intragastric administration₄、ISAP₅、ISAP₆(ii) a Two groups of controls, wherein normal saline is infused per day, after one week, three groups of experimental groups and one control group are infused with sterilized olive oil, one control group is infused with normal saline, the mice are euthanized and dissected to obtain small intestines after 20 minutes, the jejunum of the proximal duodenum segment is frozen and stained with oil red O, and hematoxylin is counterstained; the oil red O positive area was quantified by ImageJ software under double blind conditions and the ratio of this area to the total area of small intestine villi was calculated. Statistical analysis: compared to saline + olive oil. P<0.001.N＝3。

Detailed Description

Definition of

The term "polypeptide that modulates energy metabolism," also referred to as "Insulin Secretion Association Peptide (ISAP)" as used herein, refers to polypeptides derived from or derived from osteocalcin and capable of modulating energy metabolism and variants thereof.

The term "conservative amino acid substitution" as used herein refers to a substitution of the original amino acid sequence with another amino acid residue having similar properties. For example, lysine residues, arginine residues, and histidine residues are similar in having basic side chains. In addition, aspartic acid residues and glutamic acid residues are similar in having acidic side chains. Further, asparagine residues, glutamine residues, serine residues, threonine residues, tyrosine residues, and cysteine residues are similar in having uncharged polar side chains, and glycine residues, alanine residues, valine residues, leucine residues, isoleucine residues, proline residues, tryptophan residues, phenylalanine residues, and methionine residues are similar in having nonpolar side chains. In addition, tyrosine residues, phenylalanine residues, tryptophan residues, and histidine residues are similar in having aromatic side chains. Thus, it will be apparent to those skilled in the art that amino acid substitutions made in a group of amino acids having similar properties as described above do not result in any change in the properties.

The amino acid abbreviations and Chinese names used herein are as follows

The term "disease associated with abnormal fat metabolism" as used herein refers to a disease or its complications characterized by disorder of fat metabolism, which is caused by heredity or environment or both, such as, but not limited to, obesity, type II diabetes, non-alcoholic fatty liver disease, insulin resistance, hypertriglyceridemia, hypercholesterolemia, atherosclerosis, coronary heart disease.

The term "non-alcoholic fatty liver" as used herein refers to the clinical pathological syndrome characterized by excessive deposition of fat in liver cells due to factors other than alcohol.

The term "insulin resistance" as used herein refers to a state in which a cell cannot burn glucose efficiently due to a decrease in the function of insulin for lowering blood glucose. When insulin resistance is high, the human body produces excessive amounts of insulin, resulting in hypertension, dyslipidemia, heart disease, diabetes, and the like. Especially in the case of type II diabetes, insulin does not work because muscle and adipose tissue cannot recognize the increase in insulin.

As used herein, terms not explicitly defined have the meanings that are commonly understood by those skilled in the art.

Detailed description of the preferred embodiments

DMEM used in the present invention was purchased from Sigma supplemented with 10% FBS (fetal bovine serum), 1% non-essential amino acids, 1g glucose, 0.75g sodium bicarbonate, 0.1g bovine serum albumin and 1.5ml HEPES (4-hydroxyethylpiperazineethanesulfonic acid) per 500ml of medium.

3T3L1 was purchased from ATCC.

ISAP₁The sequence is Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr (SEQ ID NO. 18). ISAP₂The sequence of (A) is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Pro-Ser-Val-Pro-Tyr-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 2). ISAP₃The sequence of (a) is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Pro-Val-Pro-Tyr-Pro-Asp-Pro-Leu-Glu-Pro-Arg (SEQ ID NO. 3). ISAP₄The sequence is Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 15). ISAP₅The sequence is Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 16). ISAP₆The sequence is Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 14).

All animal experiments were approved by the animal ethics committee of the advanced technology research institute of the chinese academy of sciences, and were performed according to the requirements of the ethics committee.

Example 1

ISAP₁Functional study of

1. Establishment of obesity and non-alcoholic fatty liver model by high-fat feeding

Healthy 6-week-old male C57BL/6 SPF-rated mice were purchased from the center of laboratory animals, Guangdong province, with a body mass of 18-22 g. Divided into two groups, and the two groups are bred in SPF-level animal houses of Shenzhen advanced research institute. Control group: mice were given Normal Diet (ND) (normal diet: fat, 5%; carbohydrate, 53%; protein, 23%; total calories 25J/g), ad libitum and water, and fed for 12 weeks. Experimental groups: mice were given High Fat Diet (HFD) (high fat diet D12451, Research Diets, Inc.) and were fed free to eat and drink water for 12 weeks. Detecting the physiological index. Mice fed high fat diet weighed more than 40g, and the obesity and non-alcoholic fatty liver disease model (DIO-NAFLD) was considered to be successfully constructed. The body weight of the mice under construction is compared with that of the control group shown in FIG. 1, and the arrow indicates the time point at which the high fat diet starts to be fed.

2. Intraperitoneal injection of ISAP₁Effect on DIO-NAFLD mice

2.1 intraperitoneal injection of ISAP₁Influence on epididymal fat pad

When the weight of mice fed with the high-fat feed exceeds 40g and the blood sugar is higher than 10mMol, the ISAP of DIO-NAFLD mice for 6 weeks is carried out₁Intraperitoneal injection, 6 mice per group, and ISAP₁Dissolved in 0.01% BSA physiological saline solution, and injected once daily at 20pmol/g, 2pmol/g abdominal cavity into DIO-NAFLD mice, OCN (mouse osteocalcin protein) and ISAP₁Similarly, the concentration was 6 pmol/g. After 6 weeks, 95% CO was administered₂Euthanasia is carried out on the mouse, epididymal fat pad tissues are collected and weighed; paraffin sections were then made and the adipocyte area was observed by microscopy.

The results are shown in FIG. 2, (A) the overall appearance of the fat in mice of ND control group, HFD control group and daily intraperitoneal injection of polypeptide, compared with HFD control group, administered ISAP₁The fat tissue is obviously reduced; (B) average epididymal fat pad mass for each group of mice, and ISAP administration compared to HFD control group₁The weight of the epididymis fat pad is obviously reduced; (C) fat pad of epididymis H&E staining, revealing administration of ISAP compared to HFD control group₁The fat cells are obviously reduced; (D) according to H under double blind conditions&E stained sections calculation of average individual adipocyte surface area, indicating ISAP₁The fat cells are obviously reduced. Statistical analysis: and comparing with an HFD control group. P<0.05,**:P<0.01,***:P<0.001。

2.2 intraperitoneal injection of ISAP₁Effects on the liver

In the step 2.1, the liver is collected while the adipose tissue is collected, the appearance is photographed, then a part of the liver tissue is taken for frozen section, microscopic observation is carried out after oil red O staining, then quantitative analysis is carried out on the same area, and the experimental result is shown in figure 3. (A) Representative image of liver appearance of each group of mice, wherein lighter color indicates higher fat content; HFD + vehicle causes significant liver fat accumulation, i.e., intraperitoneal ISAP injection₁Significantly reduces the fat accumulation of the liver (B) the liver H of each group of mice&E staining, light color representing intracellular stained fat; (C) the surface area of accumulated non-alcoholic fatty liver cells obtained under double-blind conditions after oil red O staining of the liver of each group of mice was found in the liver, ISAP₁The proportion of fat was significantly reduced compared to the HFD control, indicating ISAP₁The fat content in liver cells is reduced.

2.3 intraperitoneal injection of ISAP₁The influence on the liver function and the blood fat of the mice.

In step 2.1, prior to sacrifice, the tail vein was bled and alanine Aminotransferase (ALT), alkaline phosphatase (ALP), aspartate Aminotransferase (AST), serum cholesterol, and Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL) were tested using a roche glucometer (model cobas8000) according to the manufacturer's instructions. See FIG. 4 for experiments and results, ISAP compared to HFD control, even at a dose of 2pmol/g₁It is effective in reducing ALT, ALP, AST, cholesterol and LDL.

From the above results, it can be seen that ISAP₁The intraperitoneal injection can obviously influence the fat metabolism in a mouse body, reduce the fat accumulation in liver cells and fat cells, improve the fat metabolism and effectively relieve the progress of the non-alcoholic fatty liver.

3、ISAP₁Binding to human GPRC6A

3.1 overexpression of hGPRC6A in Hela cells

1) Cell plating: hela cell suspension at 1.6X 10⁵Density of/mL plated in 6 well plates, 2mL of mem medium per well, at 37 c,5% CO₂And culturing for 24 hours.

2) The hGPRC6 overexpression vector (pReceiver-M61) was transfected into Hela cells using Lipofectamine2000(Invitrogen) according to the manufacturer's instructions and the normal medium was changed 4 hours after transfection.

3) 48 hours after transfection, the medium was discarded, the plate-bottom cells were washed twice with sterile PBS, 300. mu.L of Trizol solution was added, cellular RNA was extracted according to the instructions of RNAioso Plus (TaKaRa), treated with DNase, and then SuperScript was added^T(Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. The fluorescent PCR products at each time point were subjected to real-time monitoring analysis (Light Cycler Roche, Germany) using cDNA as a template to detect the expression level of hGPRC 6A. If necessary, puromycin is used for screening to obtain a stable expression cell strain, then qPCR is used for detecting the gene expression, and the experimental result is shown in FIG. 6A, and human GPRC6A is stably expressed in Hela in a large amount.

3.2 binding experiments of different polypeptides of OCN to cell membranes of Hela cells overexpressing hGPRC6A

The results are shown in FIG. 6B, ISAP compared to OCN₁Almost consistent membrane binding capacity, while OCN-22 bound GPRC6A but 10-fold less affinity, demonstrating ISAP₁Is the core domain of OCN, which interacts with the receptor hGPRC 6A.

4、ISAP₁Effect of acute gavage on Small intestinal fat absorption in Normal mice

The 6-week-old male wild type C57BL/6 mice were used in four groups of 3 mice each, and 200. mu.L (Sigma) of sterilized olive oil solution was injected into the intestinal tract, 30 minutes later, and OCN and ISAP were intraperitoneally injected₁(concentration 6pmol/g) and at 30 minutes the mice were sacrificed and small intestine samples were collected. Samples are from duodenum to cecum, and 3 sections with equal length are taken. After the segment near the duodenum was washed with a pre-cooled physiological saline solution, cryo-sectioning was performed, and fat absorption was observed by staining with oil red O.

The results of the experiment are shown in FIG. 5; (A) freezing and slicing jejunum of the proximal duodenum segment, and staining with oil red O and hematoxylin counterstaining; (B) for oil red O positive area under double blind condition, proceed through ImageJ softwareThe line was quantified and the ratio of this area to the total area of the small intestine villi was calculated. It can be seen that ISAP relative to saline control₁The olive oil absorption is effectively reduced, and the effect is even more obvious than that of OCN.

5. Intragastric administration of ISAP₁Effect on HFD mice

6-week-old male wild-type C57BL/6 mice were divided into 6 groups of 6 mice, and Normal Diet (ND) was given to group 1, High Fat Diet (HFD) was given to groups 2-6, control was given to group 2, and OCN and ISAP were administered to groups 3-6 by intragastric gavage at 2pmol/g body weight per day₁、ISAP₂、ISAP₃Body weight was monitored weekly for seven weeks. The results are shown in FIG. 7A, from which it is apparent that ISAP is present in comparison with the HFD control group₁The group showed significant weight loss. P<0.05,**:P<0.01,***:P<0.001。

Collecting feces of mice in 7 weeks, baking at 60 deg.C for 3 days to ensure oven drying, collecting 1 mg, soaking in 1ml mixed solution of chloroform and methanol 2: 1, pulverizing the feces with tissue homogenizer, centrifuging, collecting supernatant, and detecting triglyceride with Roche blood biochemical analyzer (see FIG. 7B). P<0.01,***:P<0.001. ISAP compared to HFD control group₁The group feces contained significantly elevated levels of triglycerides indicating intragastric ISAP administration₁Significantly reducing the absorption of triglycerides in the intestinal tract.

Example 2

ISAP₂Functional study of

1、ISAP₂Binding to human GPRC6A

1.1 overexpression of hGPRC6A in Hela cells

1) Cell plating: hela cell suspension at 1.6X 10⁵Perml density plated in 6 well plates, 2mL of LDMEM medium per well, 5% CO at 37 ℃₂And culturing for 24 hours.

3) 48 hours after transfectionThe medium was discarded, the cells at the bottom of the plate were washed twice with sterile PBS, 300. mu.L of Trizol solution was added, cellular RNA was extracted according to the instructions of RNAioso Plus (TaKaRa), treated with DNase, and then SuperScript was added^T(Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. The fluorescent PCR products at each time point were subjected to real-time monitoring analysis (Light Cycler Roche, Germany) using cDNA as a template to detect the expression level of hGPRC 6A. If necessary, puromycin is used for screening to obtain a stable expression cell strain, then qPCR is used for detecting the gene expression, and the experimental result is shown in FIG. 6A, and human GPRC6A is stably expressed in Hela in a large amount.

2、ISAP₂And binding experiment of hOCN to cell membrane of Hela cell overexpressing hGPRC6A

The experimental procedure is essentially the same as described in the examples, and the results are shown in FIG. 6B, ISAP compared to hOCN₂Having almost uniform membrane binding capacity, while hOCN-22 did not, combined with the results of item 3.2 in example 1, demonstrate ISAP₁And ISAP₂The core domains of OCN and hOCN, respectively, interact with the receptor hGPRC6A, suggesting ISAP₁And ISAP₂With the same function, subsequent signaling and biological events are caused by hgrc 6A.

2.3 OCN, hOCN22, ISAP labelled with Cy5₂Internalization of GPRC6A was promoted in Hela cells overexpressing GPRC 6A.

The cell suspension expressing hGPRC6AHela is divided into 1.6X 10⁵Per mL density was plated in 24-well plates pre-loaded with gelatin-coated coverslips, 0.5mL DMEM per well, 5% CO at 37 ℃₂And culturing for 24 hours. Before treatment, cells were starved for 4h in serum-free medium, and 100nM of Cy5-OCN, Cy5-hOCN22, Cy5-ISAP was added to each well₂Incubation at 37 ℃ for 30 min, fixation of cells using poly-methanol for 30 min, and incubation with Triton X-100(sigma) for 10 min, DAPI (Sigma) for 10 sec according to the manufacturer's instructions, cells and staining, visualization using a fluorescence confocal microscope and photography, see FIG. 6C for experimental results. As can be seen, Cy5-OCN and Cy5-ISAP₂Has distribution in the interior of the cell, while Cy5-hOCN22 is distributed in the exterior of the cellAgain, Cy5-OCN and Cy5-ISAP are illustrated₂Can bind to the receptor and enter into the cell through internalization, thereby playing a role in regulating energy metabolism.

2.4 intragastric administration of ISAP₂Effect on HFD mice

6 week-old male wild type C57BL/6 mice were divided into 6 groups of 6 mice, and the group 1 was fed with Normal Diet (ND), the groups 2 to 6 were fed with High Fat Diet (HFD), the group 3 to 6 were used as a control, and the experimental groups were each gavaged with OCN and ISAP at a dose of 2pmol/g body weight per day₁、ISAP₂、ISAP₃Body weight was monitored weekly for seven weeks. The results are shown in FIG. 7A, from which it is apparent that ISAP is present in comparison with the HFD control group₂The group showed significant weight loss.

Collecting feces of mice in 7 weeks, baking at 60 deg.C for 3 days to ensure oven drying, collecting 1 mg, soaking in 1ml mixed solution of chloroform and methanol 2: 1, pulverizing the feces with tissue homogenizer, centrifuging, collecting supernatant, and detecting triglyceride with Roche blood biochemical analyzer (see FIG. 7B). ISAP compared to HFD control group₂The group feces contained significantly elevated levels of triglycerides indicating intragastric ISAP administration₂Significantly reducing the absorption of triglycerides in the intestinal tract.

Example 3

ISAP₃Functional study of

1. Intragastric administration of ISAP₃Effect on HFD mice

6 week-old male wild type C57BL/6 mice were divided into 6 groups of 6 mice, and the group 1 was fed with Normal Diet (ND), the groups 2 to 6 were fed with High Fat Diet (HFD), the group 3 to 6 were used as a control, and the experimental groups were each gavaged with OCN and ISAP at a dose of 2pmol/g body weight per day₁、ISAP₂、ISAP₃Body weight was monitored weekly for seven weeks. The results are shown in FIG. 7A, from which it is apparent that ISAP is present in comparison with the HFD control group₃The group showed significant weight loss.

Collecting feces of mice at 7 weeks, baking at 60 deg.C for 3 days to ensure dryness, collecting 1 mg, and mixing with 1ml of chloroform and methanol 2: 1Soaking, pulverizing feces with tissue homogenizer, centrifuging, collecting supernatant, and detecting triglyceride with Roche blood biochemical analyzer (see FIG. 7B). ISAP compared to HFD control group₃The group feces contained significantly elevated levels of triglycerides indicating intragastric ISAP administration₃Significantly reducing the absorption of triglycerides in the intestinal tract.

Contrast ISAP₁、ISAP₂、ISAP₃The sequence of (A) can be known: with respect to ISAP₁,ISAP₂Has 4 insertions, 2 substitutions, and 1 deletion; with respect to ISAP₁，ISAP₃With 4 insertions, 3 substitutions; with respect to ISAP₂，ISAP₁Has 4 deletions, 2 substitutions, and 1 insertion; in summary, it is assumed that the three polypeptides are variants of each other, and that amino acid substitutions, insertions and deletions, which are well known to the person skilled in the art, can be made on the basis of these three sequences, provided that their ability to regulate energy metabolism is not significantly reduced, for example by not more than 40%, 30%, 20%, 10%, see fig. 8 in which homologous sequences of various biological sources are aligned, wherein SEQ ID No. 2: YLYQWLGAPVPYPDPLEP, SEQ ID NO. 4: YLNNGLGAPAPYPDPLEP, SEQ ID NO. 5: YLYQWLGAPVPYPDTLEP, SEQ ID NO. 6: YLYQWLGAPVPYPDPLEP, SEQ ID NO. 7: YLDHWLGAPAPYPDPLEP, SEQ ID NO. 8: YLDPGLGAPAPYPDPLEP, SEQ ID NO. 9: YLDHGLGAPAPYPDPLEP, SEQ ID NO. 10: YLDQGLGAPAPAPDPLEP, SEQ ID NO. 11: YLDSGLGAPVPYPDPLEP, which in most cases do not differ by more than four amino acid substitutions when compared in pairs, and thus it is assumed that these sequences have similar biological functions and are also encompassed by the present invention, preferably wherein the total number of deletions, substitutions and insertions does not exceed 4, e.g., 3, 2, 1. In addition, SEQ ID NO.1 has only one amino acid residue deleted compared to SEQ ID NO.18, and thus it is inferred that SEQ ID NO.1 also has a function similar to SEQ ID NO. 18.

At the same time, with respect to ISAP₂，ISAP₁、ISAP₃Equivalent to in ISAP₂The end has 1 insertion; indicating that several amino acid residues may be added at the end of the polypeptide, provided that it does not regulate energy metabolismIs reduced significantly, e.g. by not more than 40%, 30%, 20%, 10%, preferably by not more than 5, e.g. 4, 3, 2, 1 or 0 amino acid residues terminally added.

Example 4

ISAP₄、ISAP₅、ISAP₆Function of (2)

ISAP₄、ISAP₅、ISAP₆Effect of acute gavage on Small intestinal fat absorption in Normal mice

15 male wild type C57BL/6 mice at 6 weeks of age were used and divided into 5 groups of 3 mice each and three experimental groups, and ISAP was administered by gavage at 2pmol/g body weight₄、ISAP₅、ISAP₆(ii) a Two groups of controls were administered by intragastric administration of an equal volume of saline daily for one week, 8 days, and ISAP was administered by intragastric administration separately₄、ISAP₅、ISAP₆And 30 minutes after the normal saline, 200 mu L of sterilized olive oil is applied to three groups of experimental groups and a control group by intragastric administration, 200 mu L of normal saline is applied to a control group by intragastric administration, and 95% CO is applied after 50 minutes₂Euthanasia mice and dissect to obtain small intestine, and cryosection the jejunum of the proximal duodenum segment and stain with oil red O, and counterstain with hematoxylin; the oil red O positive area was quantified by ImageJ software under double blind conditions and the ratio of this area to the total area of small intestine villi was calculated. Statistical analysis: compared to saline + olive oil. P<0.001.N＝3。

The experimental results are shown in FIG. 9; the oil red O positive area was quantified by ImageJ software under double blind conditions and the ratio of this area to the total area of small intestine villi was calculated. It can be seen that ISAP relative to saline control₄、ISAP₅、ISAP₆Very effective in reducing olive oil absorption, proving them and ISAP₁、ISAP₂、ISAP₃Biologically functional equivalence, and, with reference to the sequence alignment of FIG. 8, it can also be seen that the sequence of SEQ ID NO. 12: PVPYPDPLEP and SEQ ID NO. 15: SVPSPDPLEP, SEQ IDNO.13: PYPDPLEP to SEQ ID NO. 16: the PSPDPLEP has very high similarity in sequence and is also a very conserved sequence among various species, so that the PSPDPLEP has similar biological activityThus, it is believed that these polypeptides and variants thereof can also be affected by the absorption and metabolism of fat by oral administration, i.e. they can be subjected to amino acid substitutions, insertions and deletions as are well known to the skilled person, provided that their ability to modulate energy metabolism is not significantly reduced, e.g. by not more than 40%, 30%, 20%, 10%. At the same time, ISAP₄、ISAP₅、ISAP₆Shorter and therefore less costly, and more stable, and has great potential for use as a drug for the treatment of diseases associated with abnormal fat metabolism.

All publications and patents mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalent scheme

While specific embodiments of the invention have been specifically disclosed herein, the foregoing description is intended to be illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the appended claims. The full scope of the invention should be determined by reference to the claims, their full scope of equivalents, and this specification and such variations.

Sequence listing

<110> Shenzhen advanced technology research institute

<120> polypeptide for regulating energy metabolism and use thereof

<160>18

<170>PatentIn version 3.3

<210>1

<211>14

<212>PRT

<213> mouse (Mus musculus)

<400>1

Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro

1 5 10

<210>2

<211>18

<212>PRT

<213> human (Homo sapiens)

<400>2

Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>3

<211>19

<212>PRT

<213> Artificial sequence

<220>

<223>ISAP3

<400>3

Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro Arg

<210>4

<211>18

<212>PRT

<213> rat (Rattus norvegicus)

<400>4

Tyr Leu Asn Asn Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>5

<211>18

<212>PRT

<213> bonobo (Pan trogloytes)

<400>5

Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Thr Leu

1 5 10 15

Glu Pro

<210>6

<211>18

<212>PRT

<213> Kiwi berry (Macaca mulatta)

<400>6

Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>7

<211>18

<212>PRT

<213> cattle (Bos taurus)

<400>7

Tyr Leu Asp His Trp Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>8

<211>18

<212>PRT

<213> sheep (Ovis aries)

<400>8

Tyr Leu Asp Pro Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>9

<211>18

<212>PRT

<213> wild boar (Sus scrofa)

<400>9

Tyr Leu Asp His Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>10

<211>18

<212>PRT

<213> nudease zokor (heterocarphalus glaber)

<400>10

Tyr Leu Asp Gln Gly Leu Gly Ala Pro Ala Pro Ala Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>11

<211>18

<212>PRT

<213> dog (Canis lupus)

<400>11

Tyr Leu Asp Ser Gly Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu

1 5 10 15

Glu Pro

<210>12

<211>10

<212>PRT

<213> human

<400>12

Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro

1 5 10

<210>13

<211>8

<212>PRT

<213> human

<400>13

Pro Tyr Pro Asp Pro Leu Glu Pro

1 5

<210>14

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223>IASP6

<400>14

Pro Asp Pro Leu Glu Pro

1 5

<210>15

<211>10

<212>PRT

<213> mice

<400>15

Ser Val Pro Ser Pro Asp Pro Leu Glu Pro

1 5 10

<210>16

<211>8

<212>PRT

<213> mice

<400>16

Pro Ser Pro Asp Pro Leu Glu Pro

1 5

<210>17

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> a general formula of ISAP

<220>

<221>misc_feature

<222>(3)..(3)

<223> Xaa is Tyr, Asn, Asp or absent

<220>

<221>misc_feature

<222>(4)..(4)

<223> Xaa is Gln, Asn, His, Pro, Ser or absent

<220>

<221>misc_feature

<222>(5)..(5)

<223> Xaa is Trp, Gly or absent

<220>

<221>misc_feature

<222>(6)..(6)

<223> Xaa is Leu or absent

<220>

<221>misc_feature

<222>(9)..(9)

<223> Xaa is Pro or Ser

<220>

<221>misc_feature

<222>(10)..(10)

<223> Xaa is Ala or Val

<220>

<221>misc_feature

<222>(10)..(10)

<223> Xaa is Tyr or Ser

<220>

<221>misc_feature

<222>(10)..(10)

<223> is Thr or Pro

<400>21

Tyr Leu Xaa Xaa Xaa Xaa Gly Ala Xaa Xaa Pro Xaa Pro Asp Xaa Leu

1 5 10 15

Glu Pro

<210>18

<211>15

<212>PRT

<213> mouse (Mus musculus)

<400>18

Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr

1 5 10 15

Claims

1. A polypeptide that modulates energy metabolism, said polypeptide being YLGASVPSPDPLEPT;

or SEQ ID NO. 2: YLYQWLGAPVPYPDPLEP, respectively;

or SEQ ID NO. 15: SVPSPDPLEP, respectively;

or SEQ ID NO. 16: PSPDPLEP;

or SEQ ID NO. 14: PDPLEP.

2. A pharmaceutically acceptable salt of the polypeptide of claim 1.

3. A polynucleotide encoding the polypeptide of claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell transfected with the vector of claim 4 and capable of producing the polypeptide of claim 1 under conditions in which the protein is expressible.

6. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2.

7. Use of the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2 or the pharmaceutical composition of claim 6 in the manufacture of a medicament for the treatment of a disease associated with abnormal fat metabolism; wherein the disease is a disease that may benefit from reduced fat absorption, reduced blood lipids, increased fat consumption or reduced fat accumulation.

8. The polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2 or the pharmaceutical composition of claim 6, characterized by being orally administrable.

9. Use of the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2 for the manufacture of a health product for weight loss.

10. A health product for reducing body weight comprising an effective amount of the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2.