CN111450233B

CN111450233B - Application of microbial metabolite in relieving glycolipid metabolic disorder

Info

Publication number: CN111450233B
Application number: CN202010196169.4A
Authority: CN
Inventors: 武振龙; 陈菁青; 杨鹰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2021-04-13
Anticipated expiration: 2040-03-19
Also published as: CN111450233A

Abstract

The invention relates to application of a microbial metabolite in relieving glycolipid metabolic disorder. More particularly, the present invention relates to the use of an AMP protein for preventing and/or treating obesity, fatty liver, diabetes and hyperlipidemia. The invention can effectively reduce the body weight gain and the blood fat content induced by high fat, improve the glucose tolerance, reduce the deposition of subcutaneous fat and visceral fat, relieve liver injury and liver steatosis, has important significance for relieving obesity and fatty liver and has wide application prospect.

Description

Application of microbial metabolite in relieving glycolipid metabolic disorder

Technical Field

The present invention relates to the field of biology. Specifically, the invention relates to application of a microbial metabolite, namely AMP protein in alleviating fat deposition, improving glucose tolerance and improving liver steatosis capability. More specifically, the present invention relates to the use of the microbial metabolite AMP protein in alleviating fat deposition, increasing glucose tolerance and improving hepatic steatosis, and in alleviating weight gain, increasing glucose tolerance and improving hepatic steatosis.

Background

Over the past several decades, obesity has been increasing in incidence globally. Over 6 million adults and 1 million children worldwide are reported to be obese. Overweight and obese individuals are susceptible to a range of diseases, such as type 2 diabetes, cardiovascular disease, cancer and other metabolic disorders that are metabolically abnormal, and obesity has become a major problem in the public health field that affects human health.

The occurrence of obesity is closely related to genetic factors and environmental factors, wherein intestinal microorganisms play an important role in regulating host metabolism and body inflammation. Intestinal flora disturbance is closely related to the occurrence of metabolic syndrome. Numerous studies have shown that the dietary habits of animals and humans greatly influence the composition and function of the intestinal flora and may ultimately lead to or prevent the occurrence of metabolic diseases. Probiotics are defined as active microorganisms that confer a benefit on the health of the host and, when administered in sufficient amounts, may be involved in regulating the metabolism and immunoregulatory functions of the host.

Obesity has become a major health threat problem, but at present, natural products without side effects and capable of effectively relieving obesity and related metabolic diseases are still to be developed. The Akkermansia Muciniphila (AM) strain is a novel second-generation probiotic with weight-reducing effect discovered in recent years, but is inconvenient to popularize in production due to rigor anaerobic property and culture regulation.

Therefore, there is a need in the art for an alternative to existing products that is effective in alleviating obesity and related metabolic disorders without producing side effects.

Disclosure of Invention

As described above, there is a need in the art for an alternative to existing products that is effective in alleviating obesity and related metabolic diseases without causing side effects.

The inventor discovers for the first time through previous researches that: the metabolite AMP protein of AM strain is a possible mechanism for AM bacteria to play a role in weight loss. Therefore, the present inventors have obtained AMP protein through prokaryotic expression and purification, and have studied the effects of AMP protein of various concentrations in alleviating mouse weight gain and excessive body fat deposition, improving glucose tolerance and improving liver steatosis through a high fat-induced obese mouse model, thereby completing the present invention.

Accordingly, in a first aspect of the present invention, there is provided a use of an AMP protein having a sequence of SEQ ID NO. 1 for the manufacture of a medicament for the prevention and/or treatment of obesity.

In a second aspect of the present invention, there is provided a use of AMP protein in the manufacture of a medicament for the prevention and/or treatment of fatty liver, wherein the AMP protein has a sequence of SEQ ID NO. 1.

In a third aspect of the present invention, there is provided a use of AMP protein in the manufacture of a medicament for the prevention and/or treatment of diabetes, wherein the AMP protein has a sequence of SEQ ID NO. 1.

In a fourth aspect of the present invention, there is provided a use of AMP protein having a sequence of SEQ ID NO. 1 for the preparation of a medicament for preventing and/or treating hyperlipidemia.

By adopting the AMP protein, the weight gain and the excessive physical deposition of a subject can be effectively relieved, the glucose tolerance can be improved, and the hepatic steatosis can be improved.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows the effect of different concentrations of AMP protein on body weight in high fat-induced mice; wherein, A: effect of different concentrations of AMP protein on high fat induced body weight gain in mice; b: effect of different concentrations of AMP protein on body shape of high fat-induced mice; data are expressed as mean ± sem; n is 15; different letters indicate significant differences between groups, P < 0.05;

FIG. 2 shows the effect of different concentrations of AMP protein on the adipose tissue weight in high fat-induced mice; wherein, A: effect of different concentrations of AMP protein on high fat-induced subcutaneous inguinal fat content in mice; b: influence of different concentrations of AMP protein on visceral epididymal fat content of high fat-induced mice; c: effect of different concentrations of AMP protein on high fat-induced perirenal fat content in mice; d: effect of different concentrations of AMP protein on the mesenteric fat content of high fat-induced mice; data are expressed as mean ± sem; n is 15; different letters indicate significant differences between groups, P < 0.05;

FIG. 3 shows the effect of high doses of AMP protein on the lipid content of high lipid-induced mice; wherein, A: effect of high dose AMP protein on serum triglyceride levels in high fat-induced mice; b: effect of high dose AMP protein on serum total cholesterol levels in high fat-induced mice; data are expressed as mean ± sem; n is 15; different letters indicate significant differences between groups, P < 0.05;

figure 4 shows the effect of high dose AMP protein on high fat induced glucose tolerance in mice; data are expressed as mean ± sem; n is 6; indicates significant difference compared to the high lipid group, P < 0.05;

FIG. 5 shows the effect of high dose AMP protein on high fat induced liver steatosis in mice; wherein, A: effect of high dose AMP protein on liver appearance in high fat-induced mice; b: effect of high dose AMP protein on high fat induced liver histomorphoftructure in mice; c: effect of high dose AMP protein on liver fat content in high fat-induced mice; the scale size was 50 microns and the data are expressed as mean ± sem; n is 15; different letters indicate significant differences between groups, P < 0.05.

Detailed Description

The AMP proteins of the present invention and their respective uses are described in more detail below. It should be noted that the summary above and the detailed description below are merely intended to specifically illustrate the present invention and are not intended to limit the invention in any way. The scope of the invention is to be determined by the appended claims without departing from the spirit and scope of the invention.

As described above, the object of the present invention is to provide a substitute for existing products, which can effectively alleviate obesity and related metabolic diseases without causing side effects.

Before specifically describing the present invention, the inventors herein point out: although the present inventors have completed the present invention by employing prokaryotically expressed AMP proteins, it is to be understood that AMP proteins obtained in any other manner can be used in the present invention. Specifically, the AMP protein may be a protein obtained by culturing an AM strain such as strain ATCC BAA-835 and isolating and purifying it therefrom, a protein obtained by prokaryotic expression as described herein, or a protein artificially synthesized. There is NO particular limitation on the source of AMP protein as long as it has the sequence shown in SEQ ID NO. 1.

Secondly, although the present invention employs the AMP protein of SEQ ID NO. 1, it is understood that any functionally equivalent variant of the AMP protein having one or more amino acid insertions, substitutions and/or deletions as compared to the AMP protein of SEQ ID NO. 1, but still having the function/effect of the AMP protein of SEQ ID NO. 1, may be used in the present invention.

By "obesity" or "obesity" is meant a condition resulting from an excessive accumulation of body fat, especially triglycerides, with a certain degree of significant overweight and an excessively thick fat layer. Excessive accumulation of fat in the body due to excessive food intake or altered metabolism of the body causes excessive weight gain and causes pathological, physiological changes or latency in the human body.

Obesity assessment can be performed in two ways as follows.

The first way is by the obesity, which is calculated by the following formula:

obesity ═ actual weight-standard weight ÷ standard weight × 100%

Among them, the degree of obesity is within. + -. 10%, which is called normal moderate. Obesity is greater than 10%, and is referred to as overweight. The degree of obesity is more than 20% -30%, and the obesity is called mild obesity. Obesity greater than 30% -50% is referred to as moderate obesity. The obesity degree is more than 50%, and the obesity is called as severe obesity. Obesity less than-10% is called emaciation. The degree of obesity is less than-20% and is called emaciation.

The second way is by Body Mass Index (BMI), which is an important Index for determining whether obesity is present or not and standard Body weight. BMI reflects mainly systemic overweight and obesity. Because the BMI is calculated by the ratio of the weight to the height, the BMI is more accurate in measuring the risk of heart disease, hypertension and the like of the body due to overweight than the BMI which is determined by the weight alone. The BMI is calculated as follows:

BMI(kg/m²) Weight (kg)/height squared

Wherein, when the BMI is less than 18.5, the BMI is light weight BMI, when the BMI is more than or equal to 18.5, the BMI is 24, the BMI is healthy weight BMI, when the BMI is more than or equal to 24, the BMI is overweight BMI, when the BMI is more than or equal to 28, the BMI is obese BMI.

By the AMP protein, the deposition of subcutaneous fat and/or visceral fat in an individual can be effectively reduced, thereby preventing and/or treating obesity. Thus, in one embodiment, obesity is prevented and/or treated by the AMP protein reducing deposition of subcutaneous fat and/or visceral fat.

The term "fatty liver" refers to a pathological condition of excessive fat accumulation in liver cells due to various causes, and is a common pathological change of liver rather than an independent disease.

Fatty liver disease seriously threatens the health of people in China, is the second most serious liver disease of viral hepatitis, has continuously increased incidence rate and is younger in attack age. Normal human liver tissue contains a small amount of fat, such as triglycerides, phospholipids, glycolipids, and cholesterol, and its weight is about 3% to 5% of the weight of the liver, and if too much fat accumulates in the liver, it exceeds 5% of the weight of the liver or when there is steatosis in more than 50% of the liver cells histologically, it is called fatty liver. The mild case has no symptoms, and the severe case has fierce illness. Generally, fatty liver belongs to reversible diseases, and the early diagnosis and timely treatment can recover the normal state.

As demonstrated in the examples section, AMPs of the present invention are useful for the prevention and/or treatment of fatty liver by reducing liver fat content, alleviating high fat induced morphological structure damage of liver and fat deposition in liver. Thus, in one embodiment, fatty liver is prevented and/or treated by alleviating the morphological structural damage of the liver and/or the deposition of fat in the liver.

By "diabetes" is meant a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia occurring in the long term of diabetes results in chronic damage to, and dysfunction of, various tissues, particularly the eyes, kidneys, heart, blood vessels, nerves.

Diabetes is largely divided into two categories, type 1 diabetes and type 2 diabetes. Type 1 diabetes refers to insulin-dependent diabetes mellitus, the cause of which is the inability of islet B cells to synthesize and secrete insulin themselves, due to cell-mediated autoimmune destruction. Multiple autoantibodies may be present in the serum at the time of onset. The symptoms of diabetes are obvious when type 1 diabetes is developed, ketosis is easy to occur, namely ketosis is prone to occur, the diabetes needs to depend on exogenous insulin to survive, and the diabetes is life-threatening once insulin treatment is stopped. Type 2 diabetes refers to non-insulin dependent diabetes mellitus. In this type of diabetes, the islet cells secrete more, less, or normal insulin, with the peak secretion moving backwards. Insulin receptors or post-receptor defects on insulin target cells play a major role in pathogenesis. Type 2 diabetes is 60% overweight or obese. The long-term excessive diet has high calorie intake, and the weight is gradually increased to cause obesity, and insulin resistance and blood sugar rise are caused after the obesity.

Thus, in one embodiment, the diabetes is type 2 diabetes.

The term "glucose tolerance" refers to the ability of the body to regulate blood glucose concentration. After eating rice, flour staple food or taking glucose, the normal people are almost completely absorbed by intestinal tracts, so that the blood sugar is increased, the insulin secretion is stimulated, the synthesis of liver glycogen is increased, the decomposition is inhibited, the output of liver glycogen is reduced, the utilization of glucose by tissues in vivo is increased, the postprandial maximum blood sugar does not exceed a certain level, and more or less blood sugar is kept in a relatively stable range when eating rice, flour staple food or taking glucose. This indicates that normal individuals have a strong tolerance to glucose, i.e., normal glucose tolerance.

Thus, in one embodiment, diabetes is prevented and/or treated by increasing glucose tolerance by said AMP protein.

By "hyperlipidemia" is meant a condition in which one or more lipids in the plasma are above the normal range due to abnormal fat metabolism or movement, and lipid insolubility or slight solubility in water must be associated with protein to exist in the form of lipoprotein, so that hyperlipidemia is often hyperlipoproteinemia, manifested as hypercholesterolemia, hypertriglyceridemia or both, and clinically classified into two categories: (1) primary, rare, inherited lipid metabolism disorder diseases; (2) secondary, it is commonly used in controlling bad diabetes, alcohol drinking, hypothyroidism, nephrotic syndrome, renal dialysis, renal transplantation, biliary tract obstruction, oral contraceptive, etc.

Thus, in one embodiment, hyperlipidemia is prevented and/or treated by reducing the amount of triglycerides and/or cholesterol in the blood.

In a further embodiment, the medicament comprises a prophylactically effective amount of AMP protein and/or a therapeutically effective amount of AMP protein.

As used herein, a "prophylactically effective amount" refers to an amount effective to prevent the development of obesity, fatty liver, hyperlipidemia, or diabetes in a subject following administration of an AMP protein of the present invention to the subject. It will be appreciated that the amount will vary with the species, age, size, sex, etc. of the subject and can be determined by one skilled in the art using routine techniques.

Similarly, as used herein, a "therapeutically effective amount" refers to an amount effective to alleviate, reduce and/or cure obesity, fatty liver, hyperlipidemia or diabetes suffered by a subject following administration of an AMP protein of the invention to the subject. Similarly, the amount will vary with the species, age, size, sex, etc. of the subject, and will also be related to the severity of the disease the subject is currently suffering from. One skilled in the art can determine an appropriate therapeutically effective amount by routine skill.

The present invention will be described in more detail and with reference to the following examples and drawings.

Examples

Test animal

In the test, 90 SPF-grade 3-week-old male C57BL/6 mice (purchased from Beijing Huafukang laboratory animal technology Co., Ltd.) are selected and bred in a mouse room at the temperature of 22 +/-1 ℃, the illumination time is 12 hours (8 to 20 points) per day, food and water are freely taken and drunk, and the formal test is started after one week adaptation.

Reagents and materials

pET-26b (+) expression vector: kanamycin-resistant, isopropyl thiogalactoside (IPTG) -activatable T7 promoter from Addgene, USA

Ni-NTA agarose purification resin: purchased from GE, USA

Brain heart infiltration medium powder, agar, tryptone, yeast extract: purchased from BD company of usa

XhoI and NdeI restriction enzymes and DNA ligation kit: from Takara, Dalian

Competent E.coli TOP10(ZC104) and TOP10 BL21(DE 3): from Union, Beijing Zhuang

Plasmid small and large extraction kit, agarose gel DNA recovery extraction kit: from Beijing Tiangen Biochemical technology Ltd

IPTG, lysozyme: from Yuasen, Shanghai

Coomassie brilliant blue dye, BCA protein assay kit: from Shanghai Biyuntian Biotech Co., Ltd

Protein sequence synthesis and sequencing: is completed by Shanghai Bioengineering Co Ltd

Serum triglyceride and total cholesterol detection kit: purchased from Nanjing institute of bioengineering

Paraformaldehyde, hematoxylin and eosin dyes: from China fir Jinqiao Biotechnology Co., Ltd, Beijing

The pellet feed for the mice: control ration, purchased from Research diet, consisted of 10% (kcal%) fat, 20% protein and 70% carbohydrate; high fat diets consisted of 60% (kcal%) fat, 20% protein and 20% carbohydrate, with feed nutrients as shown in table 1.

TABLE 1 feed Nutrition ingredients

Main instrument equipment

A multifunctional microplate reader:

m, available from Molecular Devices, Inc. of USA

Ultra-low temperature refrigerator, MLT: from Thermo corporation of the United states

Manual slicing machine: HM315 from Thermo company of USA

A digital imaging system: DP72 available from OLYMPUS, Japan

An ultrapure water system: arium Pro VF available from Sartoris, Germany

Refrigerated centrifuge, Legend micro 21R, available from Thermo corporation of USA

Blood glucose meter, One Touch Ultra Easy glucometer, available from Qiangsheng group, USA.

Test method

And E, culturing escherichia coli:

inoculating the transformed Escherichia coli suspension into a culture dish containing a specific resistant LB culture medium, culturing overnight at 37 ℃, selecting positive clones, shaking up in the liquid LB culture medium containing the specific resistance, and culturing at 37 ℃ and 220 r/min.

The molecular cloning, prokaryotic expression and protein purification method comprises the following steps:

plasmid extraction and enzyme digestion identification:

(1) the PUSP-AMP vector, which is a cloning vector containing a protein sequence and synthesized in Shanghai, is transformed into a competent state of Escherichia coli TOP10(ZC104), positive clones are selected and cultured overnight, plasmid extraction is carried out according to a plasmid miniextraction kit, and the concentration of the plasmid is determined.

(2) According to the instruction of the reagent, the water, the plasmid sample, 10 XH Buffer, NdeI enzyme and XhoI enzyme are mixed evenly in turn, centrifuged, and cut at 37 ℃ for 3H.

(3) Agarose gel electrophoresis was performed, and the band of interest was recovered and the concentration was determined.

(4) The expression vector pET-26b (+) empty plasmid strain was subjected to plasmid extraction and double digestion in the same procedure, and the linearized plasmid was recovered.

Constructing a prokaryotic expression vector:

(1) according to the instruction of the ligation kit, the gel recovered DNA sample, the linearized pET-26b (+) vector and the Solution I of the DNA ligase in the DNA ligation kit are sequentially added for centrifugation, and ligation is carried out overnight at 16 ℃.

(2) Adding Solution III in a DNA ligase kit, fully mixing and centrifuging.

(3) The ligation product pET-26b-AMP was added to a frozen and thawed TOP10 BL21(DE3) competent E.coli, mixed well and allowed to stand in ice bath for 30 min.

(4) The mixture was heat-shocked at 42 ℃ for 90s, rapidly transferred to an ice bath to be cooled for 3min, and then added with LB medium without antibiotics to be cultured at 37 ℃ for 1 h.

(5) After overnight culture, selecting positive clones, inoculating the positive clones in LB culture solution containing kanamycin resistance, culturing for 6h at 37 ℃, sucking part of the bacterial solution for sequencing comparison, preserving the rest part of the bacterial solution with 50% of glycerol, and storing in a refrigerator at-80 ℃.

IPTG induced protein prokaryotic expression:

and (3) inoculating the pET-26b-AMP strain with completely correct sequencing comparison into an LB culture medium containing kanamycin resistance according to the inoculation amount of 1%, shaking up gently, culturing at 37 ℃ for 220r/min, adding IPTG (isopropyl-beta-thiogalactoside) until the strain grows to the logarithmic growth phase to enable the final concentration to be 1mmol/L, continuing induction culture, sampling for 0 h, 1h, 2 h, 4 h, 5h, 6h and 7h respectively, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Coomassie brilliant blue staining, finding out the time point (5h) with the highest expression amount of the target protein, and carrying out subsequent expression experiments under the conditions.

AMP protein purification:

(1) the cells were resuspended in lysis buffer containing lysozyme and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) after centrifugation at 4,000g for 3min and washing the cells 3 times with PBS (5 mL lysis buffer was added per gram of cells to give a final concentration of 200. mu.g/mL lysozyme and 1mmo1/L PMSF).

(2) And (3) crushing the thallus suspension by using an ultrasonic crusher at the temperature of 4 ℃ until the suspension is basically clear.

(3) Centrifuging the crushed liquid at 4 deg.C for 30min at maximum rotation speed, collecting supernatant, and filtering with 0.22 μm filter membrane.

(4) Loading the Ni-NTA agarose purification resin into a column according to the reagent specification, washing the column by 5 times of column volume of deionized water, balancing by 5 times of column volume of lysis buffer, loading the liquid obtained in the previous step at the flow rate of 1mL/min, washing the column by 10 times of column volume of the washing buffer, eluting the column by using the elution buffer at the flow rate of 1mL/min, and retaining all the eluent.

(5) Concentrating the eluate with ultrafiltration centrifugal tube, washing the concentrate with sterilized purified water for 5 times, and concentrating the protein to about 1mg/mL with ultrafiltration centrifugal tube.

(6) And (3) performing ion exchange chromatography purification on the protein solution obtained in the previous step according to an AKAT ion exchange chromatography application instruction, collecting effluent corresponding to all ultraviolet absorption peaks, and analyzing the effluent by using SDS-PAGE electrophoresis and Coomassie brilliant blue staining technology, wherein the protein with the same size as the expected size is the purified target protein.

(7) Desalting the target protein by the same operation as the step (5).

(8) The concentration of the target protein is measured by using a BCA method, and the purified target protein is stored at-80 ℃ after being subpackaged. (lysis buffer formulation: 50mM NaH₂PO₄300mM NaCl, 10mM imidazole, pH 8.0; the formula of the cleaning buffer solution is as follows: 50mM NaHPO₄300mM nac1, 20mM imidazole, pH 8.0; elution buffer formulation 50mM NaHPO₄300mM nac1, 300mM imidazole, pH 8.0).

The sequencing shows that the AMP protein has a sequence shown as SEQ ID NO. 1:

SEQ ID NO:1：

MSNWITDNKP AAMVAGVGLL LFLGLSATGY IVNSKRSELD KKISIAAKEI KSANAAEITP SRSSNEELEK ELNRYAKAVG SLETAYKPFL ASSALVPTTP TAFQNELKTF RDSLISSCKK KNILITDTSS WLGFQVYSTQ APSVQAASTL GFELKAINSL VNKLAECGLS KFIKVYRPQL PIETPANNPE ESDEADQAPW TPMPLEIAFQ GDRESVLKAM NAITGMQDYL FTVNSIRIRN ERMMPPPIAN PAAAKPAAAQ PATGAASLTP ADEAAAPAAP AIQQVIKPYM GKEQVFVQVS LNLVHFNQPK AQEPSED

treatment of test animals:

prior to the start of the official trial, mice were randomly divided into 6 groups of 15 mice by body weight, including:

control group (ND): feeding control daily ration;

proteome (H-AMP): feeding control ration and high dose AMP protein;

high fat group (HFD): feeding high fat diet;

high-fat and low-dose proteome (HFD-L-AMP): feeding a high fat diet and a low dose of protein;

high and medium dose proteome (HFD-M-AMP): feeding high fat diet and medium dose protein;

high fat and high dose proteome (HFD-H-AMP): feeding high fat diet and high dose of protein.

The test period is 16 weeks, AMP protein is added into daily drinking water at 1-9 weeks, from 10 weeks to 16 weeks, protein group mice are fed with AMP protein, the rest groups are fed with physiological saline of the same volume as the control, and the protein used for the mouse experiment is measured to have purity of more than 97% by HPLC.

In vitro experiments demonstrated that AMP protein was not toxic to cells. Specific protein treatment amounts and treatment modes are shown in Table 2. During the test period, the water drinking of the mice is changed every day, the feed is added every two days, and the weight of the mice is weighed and recorded every two weeks. The test was conducted according to the requirements of the laboratory animal care committee of the university of agriculture in china.

TABLE 2 mouse AMP protein intake and mode of intake

Glucose tolerance test:

one week before the end of the experiment, 6 mice were taken from the high-fat group and the high-fat high-dose AMP protein group, respectively, and subjected to an Oral Glucose Tolerance Test (OGTT). After fasting for 6 hours, each mouse was placed individually in a clean sterile mouse cage, fed with 50% glucose in an amount of 2.0g/kg body weight, and measured for tail blood glucose concentration with a blood glucose meter after 0, 15, 30, 60, and 120 minutes, respectively.

Collecting samples:

after the test was completed, the mice were fasted for 6 hours, and then were sacrificed by cervical dislocation after collecting blood of the mice by an eyeball method. Subcutaneous inguinal fat, abdominal epididymal fat, perirenal fat and mesenteric fat were isolated and collected and recorded by weighing and photographed. The isolated mouse liver tissue was fixed in 4% paraformaldehyde for 24 hours, washed 3 times with physiological saline, and stored in 70% ethanol for subsequent tissue slice preparation.

And (3) detecting the blood fat content of the serum:

the contents of serum triglyceride and total cholesterol were determined according to the kit method of Nanjing Kangji Co.

Liver tissue sectioning and staining:

preparation of liver tissue sections:

tissue dehydration, transparency and waxing: taking out the tissue from 70% ethanol, sequentially soaking in 80% ethanol for 40 min; 1h with 95% ethanol; anhydrous ethanol I for 30 min; anhydrous ethanol II for 30 min; xylene I for 5 min; xylene II for 5 min; wax solution I1 h; wax solution II for 1 h.

Embedding: the wax solution is poured into an embedding box, and after the embedding box is placed into a tissue block, a wax holder is installed.

Cutting into slices: the wax block was cut into continuous 5 μm wax strips with a manual microtome, and the wax strips were then immersed in a 43 ℃ water bath and allowed to adhere to the slide surface.

Baking slices: keeping the temperature at 37 ℃ overnight, and baking the slices at 62 ℃ for 1 h.

Hematoxylin-eosin staining:

dewaxing: sequentially immersing slices in xylene I for 10 min; xylene II for 10 min; absolute ethyl alcohol for 8 min; 95% ethanol for 5 min; 70% ethanol for 5 min; 50% ethanol for 5 min; distilled water for 5 min.

Dyeing: adding hematoxylin staining solution dropwise for staining for 1 min; washing with distilled water for 3 times, each for 2 min; differentiating with 1% hydrochloric acid alcohol solution for 5 sec; washing with distilled water for 5min for 3 times; dripping eosin dye solution for dyeing for 1 min; the distilled water was washed 3 times for 2min each time.

Thirdly, dehydrating, transparent and sealing: sequentially immersing the slices in 70% ethanol for 2 sec; 95% ethanol for 5 sec; absolute ethyl alcohol for 5 min; xylene I for 5 min; xylene II for 5 min; drop 1 drop of neutral gum onto the slide, cover the slide and dry overnight. And (5) observing and photographing under a microscope.

Data statistics

Data are presented as mean ± SEM and analyzed by one-way anova. Duncan's multiple comparisons were performed using GraphPad Prism version 7.0 (GraphPad software, san Diego, Calif.) to determine differences, with P <0.05 considered significant.

Test results

In the present invention, the inventors examined various influences of AMP protein using mice as subjects.

Statistical results of mouse body weight gain are shown in fig. 1A. From this figure, the increase in body weight of mice in the high fat group was significantly higher than that in the control group (P < 0.05); while the addition of high doses of AMP protein significantly reduced the high fat induced increase in body weight gain in mice (P < 0.05). Compared with the control group, the increase of the body weight of the mice fed with the single high-dose AMP protein tends to be reduced, and the difference does not reach a remarkable level.

The observation of the mouse body shape after the experiment is completed is shown in FIG. 1B. Compared with the control group, the mice fed with the high-fat feed are obviously fatter than the control group, and the mice fed with the high-dose AMP protein can relieve the fatness of the mice under the induction of high fat. The results show that high-dose AMP protein can remarkably reduce obesity caused by mice induced by high fat.

Statistical results of mouse subcutaneous fat weight are shown in fig. 2. The inguinal fat content of mice in the high-fat group was significantly increased (P <0.05) compared to the control group, and the low-dose AMP protein feeding had no significant effect on the increase in the inguinal fat content of the high-fat-induced mice, but the medium-dose AMP protein feeding tended to decrease the inguinal fat content of the mice, while the high-dose AMP protein significantly decreased the inguinal fat content of the high-fat-induced mice (P <0.05) (fig. 2A). Similarly, statistical analysis of epididymal fat (fig. 2B), perirenal fat (fig. 2C) and mesenteric fat weight (fig. 2D) in visceral fat of mice revealed that the low dose of AMP protein administered had no significant effect on the high fat-induced increase in adipose tissue in mice, but the medium dose of AMP protein administered tended to decrease the visceral adipose tissue increase in mice, whereas the high dose of AMP protein administered significantly decreased the high fat-induced increase in epididymal fat, perirenal fat and mesenteric fat weight (P <0.05) (fig. 2B, 2C and 2D). Feeding high doses of AMP protein alone had no significant effect on the subcutaneous inguinal fat and visceral fat content of mice compared to the control group. The result shows that the high-dose AMP protein can obviously reduce the weight of subcutaneous fat and visceral fat of a mouse induced by high fat and reduce excessive accumulation of fat, so that the mice of a high-dose protein treatment group are selected for detection in follow-up experiments.

As can be seen from fig. 3, the contents of triglyceride and total cholesterol in the serum of the mice in the high-fat group were significantly increased (P <0.05) compared to the control group (fig. 3A and 3B), while the administration of high dose of AMP protein significantly reduced the increase of triglyceride and total cholesterol in the serum of the mice induced by high fat (P <0.05) (fig. 3A and 3B). Compared with the control group, the serum content of triglyceride and total cholesterol of the mice is not obviously changed by feeding the AMP protein alone with high dose. The results show that the high-dose AMP protein can obviously reduce the content of triglyceride and total cholesterol in the serum of the mouse induced by high fat.

Obesity usually causes other chronic metabolic diseases of the body, such as type 2 diabetes, hyperlipidemia, fatty liver, and the like. To investigate the role of AMP protein in maintaining high lipid-induced glucose homeostasis in obese mice, we performed a glucose tolerance test on mice. The results show that high dose AMP protein feeding significantly increased glucose tolerance in high lipid-induced mice, and blood glucose concentrations were significantly lower than those in the high lipid group (P <0.05) both 15 and 30min after glucose gavage (fig. 4).

Long-term high concentrations of free fatty acids in the blood of obese individuals tend to deposit ectopically in the liver tissue, resulting in liver steatosis. In order to study the role of AMP protein in alleviating the fatty degeneration of liver of high fat-induced obese mice, the morphology of liver tissue of the mice was observed, and the content of liver fat was measured. Observing the livers just taken out of the mice of each group, and finding that the livers of the mice fed with the high fat group are brownish yellow, and tissues are swollen and fragile; whereas, mice fed high doses of AMP protein had normal liver morphology and bright red color (FIG. 5A). The liver cell morphology slicing result shows that the liver cells of mice in the high fat group are irregularly arranged and have more vacuole-like lipid droplets, while the liver morphology structure of the mice fed with high-dose AMP protein is consistent with that of the mice in the control group and the mice fed with AMP protein alone, the liver fat cell morphology structure is normal, and the vacuole-like lipid droplets do not appear (figure 5B). Liver triglyceride level assay results showed that high fat induction significantly increased the level of triglyceride in the liver of mice (P <0.05) compared to the control group, whereas high dose AMP protein administration significantly decreased the level of triglyceride in the liver of high fat-induced mice (P <0.05) (fig. 5C). Feeding high doses of AMP protein alone had no significant effect on hepatic triglyceride levels compared to the control group. These results indicate that high doses of AMP protein alleviate high lipid-induced impairment of liver morphological structure and fat deposition in the liver.

In conclusion, AMP protein can improve obesity caused by high fat induction, reduce deposition of subcutaneous fat and visceral fat induced by high fat, enhance glucose tolerance and relieve damage of liver morphological structure and liver steatosis. The invention not only discloses the important function of AMP protein in relieving obesity and fatty liver, but also can be popularized and applied in clinic for treating obesity and related metabolic diseases.

Sequence listing

<110> university of agriculture in China

<120> application of microbial metabolite in relieving glycolipid metabolic disorder

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 317

<212> PRT

<213> Akkermansia muciniphila

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Met Ser Asn Trp Ile Thr Asp Asn Lys Pro Ala Ala Met Val Ala Gly

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Val Gly Leu Leu Leu Phe Leu Gly Leu Ser Ala Thr Gly Tyr Ile Val

20 25 30

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

35 40 45

Glu Ile Lys Ser Ala Asn Ala Ala Glu Ile Thr Pro Ser Arg Ser Ser

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Asn Glu Glu Leu Glu Lys Glu Leu Asn Arg Tyr Ala Lys Ala Val Gly

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Ser Leu Glu Thr Ala Tyr Lys Pro Phe Leu Ala Ser Ser Ala Leu Val

85 90 95

Pro Thr Thr Pro Thr Ala Phe Gln Asn Glu Leu Lys Thr Phe Arg Asp

100 105 110

Ser Leu Ile Ser Ser Cys Lys Lys Lys Asn Ile Leu Ile Thr Asp Thr

115 120 125

Ser Ser Trp Leu Gly Phe Gln Val Tyr Ser Thr Gln Ala Pro Ser Val

130 135 140

Gln Ala Ala Ser Thr Leu Gly Phe Glu Leu Lys Ala Ile Asn Ser Leu

145 150 155 160

Val Asn Lys Leu Ala Glu Cys Gly Leu Ser Lys Phe Ile Lys Val Tyr

165 170 175

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

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Asp Glu Ala Asp Gln Ala Pro Trp Thr Pro Met Pro Leu Glu Ile Ala

195 200 205

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

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Gly Met Gln Asp Tyr Leu Phe Thr Val Asn Ser Ile Arg Ile Arg Asn

225 230 235 240

Glu Arg Met Met Pro Pro Pro Ile Ala Asn Pro Ala Ala Ala Lys Pro

245 250 255

Ala Ala Ala Gln Pro Ala Thr Gly Ala Ala Ser Leu Thr Pro Ala Asp

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Glu Ala Ala Ala Pro Ala Ala Pro Ala Ile Gln Gln Val Ile Lys Pro

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Tyr Met Gly Lys Glu Gln Val Phe Val Gln Val Ser Leu Asn Leu Val

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His Phe Asn Gln Pro Lys Ala Gln Glu Pro Ser Glu Asp

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Claims

1. Use of a protein with a sequence of SEQ ID NO. 1 in the preparation of a medicament for the prevention and/or treatment of obesity.

2. The use according to claim 1, wherein the medicament prevents and/or treats obesity by the protein reducing deposition of subcutaneous fat and/or visceral fat.

3. Application of a protein with a sequence of SEQ ID NO. 1 in preparing a medicament for preventing and/or treating fatty liver.

4. The use according to claim 3, wherein the medicament prevents and/or treats fatty liver by alleviating the morphological structural damage of liver and/or the deposition of fat in liver.

5. An application of a protein with a sequence of SEQ ID NO. 1 in preparing a medicament for preventing and/or treating diabetes.

6. The use of claim 5, wherein the diabetes is type 2 diabetes.

7. The use according to claim 5, wherein the medicament prevents and/or treats diabetes by the protein increasing glucose tolerance.

8. Application of a protein with a sequence of SEQ ID NO 1 in preparing a medicament for preventing and/or treating hyperlipidemia.

9. The use according to claim 8, wherein the medicament prevents and/or treats hyperlipidemia by reducing the amount of triglyceride and/or cholesterol in blood.

10. The use according to any one of claims 1 to 9, wherein the medicament comprises a prophylactically effective amount of a protein having the sequence of SEQ ID No. 1 and/or a therapeutically effective amount of a protein having the sequence of SEQ ID No. 1.