WO2018165936A1 - 调节能量代谢的多肽及其用途 - Google Patents

调节能量代谢的多肽及其用途 Download PDF

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WO2018165936A1
WO2018165936A1 PCT/CN2017/076886 CN2017076886W WO2018165936A1 WO 2018165936 A1 WO2018165936 A1 WO 2018165936A1 CN 2017076886 W CN2017076886 W CN 2017076886W WO 2018165936 A1 WO2018165936 A1 WO 2018165936A1
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seq
polypeptide
isap
fat
pro
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PCT/CN2017/076886
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English (en)
French (fr)
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任培根
张键
滕斌
李健
姚振宇
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深圳先进技术研究院
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Priority to KR1020197029172A priority Critical patent/KR102432962B1/ko
Priority to PCT/CN2017/076886 priority patent/WO2018165936A1/zh
Priority to EP17901101.0A priority patent/EP3626734A4/en
Priority to JP2019550656A priority patent/JP7030834B2/ja
Publication of WO2018165936A1 publication Critical patent/WO2018165936A1/zh
Priority to US16/572,584 priority patent/US11098083B2/en
Priority to US17/359,370 priority patent/US11566048B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biomedical, and in particular to polypeptides that modulate energy metabolism and their use in the manufacture of a medicament for the treatment of diseases associated with abnormalities in energy metabolism, particularly fat metabolism.
  • Obesity is a major and growing health problem worldwide. Obesity is also a risk factor for the development of many common diseases such as atherosclerosis, hypertension, type 2 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 growing in all developed countries.
  • Fatty liver refers to a lesion caused by excessive accumulation of fat in liver cells. There are many causes of fatty liver, such as alcoholism, unreasonable diet, and sedentary. Its incidence in China is increasing and it is a threat to people's health.
  • Osteocalcin is a vitamin K-dependent calcium-binding protein synthesized and secreted by osteoblasts, a non-collagen acidic glycoprotein whose vitamin K-dependent glutamate residues are osteocalcin and Ca 2 + combines important functional groups [1, 2] .
  • the present inventors have unexpectedly discovered a polypeptide capable of regulating energy metabolism derived from osteocalcin; it has functions of reducing fat absorption, reducing liver fat, blood lipids in peripheral blood, and reducing fat cell volume, and can effectively remove liver.
  • the accumulation of fat in cells, lowering blood lipid levels, and reducing body fat tissue play a role in the treatment of steatohepatitis, regulation of hypertension, and other cardiovascular diseases.
  • Some aspects of the invention relate to polypeptides that modulate energy metabolism, represented by the formula M 1 -Z a -M 2 , wherein:
  • M 1 and M 2 are each independently a polypeptide segment having no more than 5, 4, 3, 2 or 1 amino acid residues or absent;
  • Z a is Tyr-Leu-X 1 -X 2 -X 3 -X 4 -Gly-Ala-X 5 -X 6 -Pro-X 7 -Pro-Asp-X 8 -Leu-Glu-Pro, wherein:
  • X 1 is Tyr, Asn, Asp or absent
  • X 2 is Gln, Asn, His, Pro, Ser or does not exist
  • X 3 is Trp, Gly or does not exist
  • X 4 is Leu or does not exist
  • X 5 is Pro or Ser
  • X 6 is Ala or Val
  • X 7 is Tyr or Ser
  • X 8 is Thr or Pro
  • the Z a may optionally have a medium amino acid substitution, insertion or deletion 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, and most preferably does not exceed 1.
  • 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.
  • Z a is YLYQWLGAPVPYPDPLEP, M 1 is absent and M 2 is present in the polypeptide Is Arg, ie the amino acid sequence of the polypeptide is set forth in SEQ ID NO. 3; in still other embodiments, Z a is YLGASVPSPDPLEP in the polypeptide, M 1 is absent and M 2 is Thr, ie the amino acid sequence of the polypeptide As shown in SEQ ID NO.
  • polypeptides which modulate 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.
  • SEQ ID NO. 12 PVPYPDPLEP
  • SEQ ID NO. 14 PDPLEP
  • SEQ ID NO. 15 SVPSPDPLEP
  • SEQ ID NO. 16 PSPDPLEP.
  • SEQ ID NO. 12 PVPYPDPLEP
  • 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 invention relate to such a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, which has the effect of reducing fat absorption, lowering blood lipid levels, alleviating non-alcoholic fatty liver, and reducing adipose tissue in the body.
  • Some aspects of the invention are directed to polynucleotides encoding the polypeptides described above.
  • Some aspects of the invention relate to vectors comprising the polynucleotides described above.
  • Some aspects of the invention relate to host cells transfected with the aforementioned vectors and capable of producing a polypeptide of the invention under conditions in which the protein can be expressed.
  • Some aspects of the invention are directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the invention described above, or a pharmaceutically acceptable salt thereof.
  • the polypeptide described in the present invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof 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 disorder associated with an abnormality in energy metabolism, more preferably fat metabolism, which is Benefit from diseases with reduced fat absorption, decreased blood lipids, increased fat consumption, or reduced fat accumulation, such as obesity, type 2 diabetes, nonalcoholic fatty liver, insulin resistance, hypertriglyceridemia, hyperglycemia, high cholesterol, arteries Atherosclerosis, coronary heart disease.
  • diseases with reduced fat absorption, decreased blood lipids, increased fat consumption, or reduced fat accumulation such as obesity, type 2 diabetes, nonalcoholic fatty liver, insulin resistance, hypertriglyceridemia, hyperglycemia, high cholesterol, arteries Atherosclerosis, coronary heart disease.
  • Some aspects of the invention relate to methods of treating a disorder associated with abnormalities in fat metabolism comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, which is Benefit from diseases with reduced fat absorption, decreased blood lipids, increased fat consumption, or reduced fat accumulation, such as obesity, type 2 diabetes, nonalcoholic fatty liver, insulin resistance, hypertriglyceridemia, hyperglycemia, high cholesterol, arteries Atherosclerosis, coronary heart disease.
  • diseases with reduced fat absorption, decreased blood lipids, increased fat consumption, or reduced fat accumulation such as obesity, type 2 diabetes, nonalcoholic fatty liver, insulin resistance, hypertriglyceridemia, hyperglycemia, high cholesterol, arteries Atherosclerosis, coronary heart disease.
  • a polypeptide of the invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof, can be combined with any of the pharmaceutical and method compositions known for treating diseases associated with abnormalities in fat metabolism Apply.
  • a polypeptide of the invention is used to treat a disorder associated with abnormalities in fat metabolism, which may benefit from reduced fat absorption, decreased blood lipids, increased fat consumption, or fat Accumulated reduced diseases such as obesity, type 2 diabetes, nonalcoholic fatty liver, insulin resistance, hypertriglyceridemia, high cholesterol, atherosclerosis, coronary heart disease.
  • a disorder associated with abnormalities in fat metabolism which may benefit from reduced fat absorption, decreased blood lipids, increased fat consumption, or fat Accumulated reduced diseases such as obesity, type 2 diabetes, nonalcoholic 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 weight loss.
  • Some aspects of the invention relate to a health care product for weight loss comprising a polypeptide of the invention or a pharmaceutically acceptable salt thereof.
  • a polypeptide of the invention can be administered by a variety of conventional means, preferably orally.
  • the polypeptide of the present invention can have a direct effect on the absorption of fat. Oral administration reduces the inconvenience of conventional polypeptide administration, has few peptide residues, greatly reduces the cost, and has a very significant potential advantage as a drug.
  • Figure 1 shows the weight comparison of Diet-Induced-Obesity & non-alcoholic fatty liver disease (DIO-NAFLD) with normal controls after 12 weeks of feeding C57BL/6 mice with high fat diet; Normal diet (ND) and high fat diet (HFD).
  • DIO-NAFLD Diet-Induced-Obesity & non-alcoholic fatty liver disease
  • Figure 2 shows the injection of different concentrations of ISAP 1 and OCN (mouse osteocalcin) in DIO-NAFLD mice fed a high-fat diet for 6 weeks, compared with the control group (high fat diet mice only).
  • the effect of ISAP 1 on mice (A) changes in the epididymal fat pad of the ND control group, the HFD control group, and the daily intraperitoneal injection of OCN or ISAP 1 group; (B) the average mass of the epididymal fat pad in each group; (C) the epididymal fat pad tissue sections of each group Hematoxylin and eosin (H&E) staining; (D) Average fat cell surface area calculated from H&E stained sections under double-blind conditions.
  • Statistical analysis Comparison with the HFD control group. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • Figure 3 shows intraperitoneal injection of ISAP 1 pair after injection of different concentrations of ISAP 1 and OCN in DIO-NAFLD mice fed a high-fat diet for 6 consecutive weeks compared to the control group (high fat diet mice only) The effect of mouse liver.
  • A Representative images of the liver appearance of each group of mice;
  • B H&E staining of liver tissue sections of each group of mice;
  • C Accumulation of non-obtained conditions obtained by liver oil red O staining in each group of mice The surface area of alcoholic fatty liver cells.
  • Statistical analysis for comparison with the HFD control group. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001. There were 6 mice in each group for analysis.
  • Figure 4 shows the injection of ISAP 1 on liver function in mice after injection of different concentrations of ISAP 1 and OCN in DIO-NAFLD mice fed a high-fat diet for 6 weeks compared with the control group (high fat diet mice only). And the effects of blood lipids.
  • A alanine aminotransferase (ALT);
  • B alkaline phosphatase (ALP);
  • C aspartate aminotransferase (AST);
  • D serum total cholesterol (TC);
  • E low density lipoprotein (LDL)
  • F High density lipoprotein
  • Statistical analysis *: Comparison with the HFD control group. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • Figure 5 shows the effect of intragastric administration of ISAP 1 and OCN on intestinal fat absorption.
  • ND-fed wild-type C57BL/6 mice were intragastrically sterilized with olive oil (30 minutes), and the mice were euthanized 30 minutes after intraperitoneal injection of ISAP 1 and OCN, and the small intestine was dissected to freeze the jejunum of the duodenum.
  • Sections were stained with Oil Red O and counterstained with hematoxylin (A); the oil red O-positive area under double-blind conditions was quantified by ImageJ software and the ratio of this area to the total area of small intestine villi was calculated (B).
  • Statistical analysis compared with saline + olive oil group. ***: p ⁇ 0.001.
  • N 3.
  • Figure 6 shows the binding and receptor internalization of ISAP 1 and ISAP 2 to human GPRC6A, respectively.
  • A Human GPRC6A overexpresses in HeLa cells;
  • B ISAP 1 , OCN and OCN-22; and binding experiments of ISAP 2 , hOCN and hOCN-22 with huGPRC6A overexpressing HeLa cells;
  • C band Cy-5-labeled OCN (Cy5-OCN), Cy-5-labeled OCN-22 (Cy5-Ocn22), Cy-5-labeled ISAP 2 (Cy5-ISAP 2 ) overexpressed in GPRC6A The effect of cells on the internalization of GPRC6A.
  • Figure 7 shows the effect on mice after seven weeks after intragastric administration of OCN, ISAP 1 , ISAP 2 , ISAP 3 : (A) changes in body weight of mice; (B) content of triglycerides in mouse feces Comparison.
  • Figure 8 shows a comparison of the sequences of ISAP from different species.
  • Figure 9 shows the effect of intragastric administration of ISAP 4 , ISAP 5 , and ISAP 6 on intestinal fat absorption.
  • Fifteen wild-type C57/b mice fed ND were divided into 5 groups, 3 rats in each group, and 3 experimental groups were administered with ISAP 4 , ISAP 5 and ISAP 6 respectively .
  • Two groups of controls were administered daily. After administration of physiological saline for one week, three groups of experimental groups and one control group were intragastrically administered with sterilized olive oil, and one control group was intragastrically administered with physiological saline. After 20 minutes, the mice were euthanized and dissected to obtain the small intestine, near the duodenum.
  • the jejunum was frozen and sectioned and stained with oil red O, and hematoxylin was counterstained; the oil red O-positive area was quantified by ImageJ software under double-blind conditions and the ratio of the area to the total area of the small intestine was calculated.
  • energy-modulating polypeptide also referred to herein as "insulin secretion association Peptide (ISAP)” as used herein refers to a polypeptide derived from osteocalcin or derived from osteocalcin and capable of regulating energy metabolism and Its variant.
  • conservative amino acid substitution refers to the replacement of the original amino acid sequence with another amino acid residue having similar properties.
  • lysine residues, arginine residues, and histidine residues are similar in that they have a basic side chain.
  • aspartic acid residues and glutamic acid residues are similar in that they have acidic side chains.
  • asparagine residues, glutamine residues, serine residues, threonine residues, tyrosine residues and cysteine residues are similar in that they have uncharged polar side chains
  • a glycine residue, an alanine residue, a proline residue, a leucine residue, an isoleucine residue, a proline residue, a tryptophan residue, a phenylalanine residue, and a Thionine residues are similar in that they have non-polar side chains.
  • the tyrosine residue, the phenylalanine residue, the tryptophan residue, and the histidine residue are similar in having an aromatic side chain.
  • disease associated with abnormal fat metabolism refers to a disease characterized by a disorder of fat metabolism or a complication thereof, such as, but not limited to, obesity, type II diabetes, which is caused by genetic or environmental or both.
  • non-alcoholic fatty liver refers to a clinicopathic syndrome characterized by excessive deposition of fat in hepatocytes due to factors other than alcohol.
  • insulin resistance refers to a state in which cells cannot effectively burn glucose due to a decrease in the function of insulin for lowering blood sugar.
  • insulin resistance is high, the human body Excessive insulin is produced, leading to hypertension, abnormal lipidemia, heart disease and diabetes.
  • insulin does not work because muscle and adipose tissue cannot recognize the increase in insulin.
  • the DMEM used in the present invention was purchased from Sigma, supplemented with 10% FBS (fetal calf serum), 1% non-essential amino acid, 1 g glucose, 0.75 g sodium bicarbonate, 0.1 g bovine serum albumin and 1.5 per 500 ml of the medium.
  • FBS fetal calf serum
  • Ml HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • 3T3L1 is purchased from ATCC.
  • the ISAP 1 sequence is Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr (SEQ ID NO. 18).
  • the sequence of ISAP 2 is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 2).
  • the sequence of ISAP 3 is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg (SEQ ID NO. 3).
  • the ISAP 4 sequence is Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 15).
  • the ISAP 5 sequence was Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 16).
  • the ISAP 6 sequence was Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 14).
  • Healthy 6-week-old male C57BL/6SPF mice were purchased from the Experimental Animal Center of Guangdong province with a body mass of 18-22 g. Divided into two groups, raised in the SPF animal room of Shenzhen Advanced Research Institute.
  • Control group Normal feed (ND) was administered to mice (normal feed: fat, 5%; carbohydrate, 53%; protein, 23%; total calories 25 J/g), free feeding and drinking, and fed for 12 weeks.
  • HFD high-fat diet
  • the comparison between the weight of the constructed mouse and the control group is shown in Figure 1.
  • the arrow indicates the time point at which the high fat diet was started.
  • High-fat diet-fed mice weighed more than 40 g and blood glucose above 10 mMol.
  • DIO-NAFLD mice were injected intraperitoneally with ISAP 1 for 6 weeks. Each group had 6 mice, and ISAP 1 was dissolved in 0.01% BSA.
  • a dose of 20 pmol/g and 2 pmol/g was intraperitoneally injected into DIO-NAFLD mice, and OCN (mouse osteocalcin protein) was similar to ISAP 1 at a concentration of 6 pmol/g.
  • OCN mouse osteocalcin protein
  • FIG. 2 The experimental results are shown in Fig. 2, (A) the overall appearance of fat in the ND control group, the HFD control group, and the daily intraperitoneal injection of the polypeptide group. Compared with the HFD control group, the administration of ISAP 1 significantly reduced the adipose tissue; The average mass of the epididymal fat pad of the mice in the group was significantly lower than that of the HFD control group by the application of ISAP 1 ; (C) H&E staining of the epididymal fat pad of each group, revealing that ISAP 1 was administered compared with the HFD control group. The fat cells were significantly reduced; (D) the average single fat cell surface area calculated from H&E stained sections under double-blind conditions, indicating that ISAP 1 significantly reduced the fat cells. Statistical analysis: Comparison with the HFD control group. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • step 2.1 the liver is collected while collecting adipose tissue, and the appearance photograph is taken. Then, part of the liver tissue is taken for frozen sectioning, and the oil red O staining is used for microscopic observation, and then the same area is quantitatively analyzed.
  • step 2.1 before the mice were sacrificed, blood was collected from the tail vein, and alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), serum were measured using a Roche blood glucose meter (model cobas8000) according to the manufacturer's instructions. Cholesterol, as well as low density lipoprotein (LDL) and high density lipoprotein (HDL). EXPERIMENTAL AND RESULTS Referring to Figure 4, even at a dose of 2 pmol/g, ISAP 1 was effective in reducing ALT, ALP, AST, cholesterol, and LDL compared to the HFD control.
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • serum serum were measured using a Roche blood glucose meter (model cobas8000) according to the manufacturer's instructions. Cholesterol, as well as low density lipoprotein (LDL) and high density
  • ISAP 1 intraperitoneal injection of ISAP 1 can significantly affect the body fat metabolism in mice, reduce fat accumulation in liver cells and fat cells, improve fat metabolism, and effectively alleviate the progression of nonalcoholic fatty liver disease. .
  • hGPRC6 overexpression vector (pReceiver-M61) was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and the normal medium was changed 4 hours after transfection.
  • mice Six-week-old male wild-type C57BL/6 mice were divided into four groups of 3 animals each, and 200 ⁇ L (Sigma) of sterilized olive oil solution was infused into the intestine. After 30 minutes, OCN and ISAP 1 were intraperitoneally injected (concentration was 6 pmol/ g), and the mice were sacrificed at 30 minutes and the small intestine samples were collected. The sample was from the duodenum to the cecum segment, taking an equal length of 3 segments. After washing with a pre-cooled physiological saline solution near the duodenum, frozen sections were taken, and fat absorption was observed using oil red O staining.
  • mice Six-week-old male wild-type C57BL/6 mice were divided into 6 groups, 6 in each group. The first group was given normal feed (ND), the second group was given high fat diet (HFD), and the second group was used as control. Groups 3-6 were experimental groups, and OCN, ISAP 1 , ISAP 2 , and ISAP 3 were administered intragastrically at 2 pmol/g body weight for 7 weeks, and body weight was monitored once a week. The results of the experiment are shown in Fig. 7A. It is apparent from the figure that a significant weight loss occurred in the ISAP 1 group relative to the HFD control group. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • the feces of the 7th week were collected and baked at 60 ° C for 3 days to ensure drying. Then, 1 mg was taken, soaked in a mixed solution of 1 ml of chloroform and methanol 2 to 1, and then the stool was pulverized by a tissue homogenizer. After taking the supernatant, the triglyceride was detected by Roche blood biochemistry instrument, and the experimental results are shown in Fig. 7B. **: P ⁇ 0.01, ***: P ⁇ 0.001. Compared with the HFD control group, the content of triglyceride in the stool of the ISAP 1 group was significantly increased, indicating that intragastric administration of ISAP 1 significantly reduced the absorption of triglyceride in the intestinal tract.
  • hGPRC6 overexpression vector (pReceiver-M61) was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and the normal medium was changed 4 hours after transfection.
  • the experimental method is basically the same as that described in the examples.
  • the experimental results are shown in Fig. 6B.
  • IASP 1 and ISAP 2 are the core domains of OCN and hOCN, respectively, which interact with the receptor hGPRC6A, and thus ICAP 1 and ISAP 2 are considered to have the same function, and subsequent signaling and biological events are caused by hGPRC6A.
  • Cy5-tagged OCN, hOCN22, ISAP 2 promotes internalization of GPRC6A in GPRC6A overexpressing HeLa cells.
  • the hGPRC6AHela cell suspension was plated at a density of 1.6 ⁇ 10 5 /mL in a 24-well plate pre-placed with gelatin-coated coverslips, 0.5 mL DMEM per well, and cultured at 37 ° C, 5% CO 2 for 24 hours. .
  • the cells were starved for 4 h in serum-free medium before treatment, and 100 nM of Cy5-OCN, Cy5-hOCN22, Cy5-ISAP 2 were added to each well, and incubated at 37 ° C for 30 minutes.
  • the cells were fixed with polymethanol for 30 minutes and added to Triton. Incubate X-100 (sigma) for 10 minutes, DAPI (Sigma) for 10 seconds.
  • mice Six-week-old male wild-type C57BL/6 mice were divided into 6 groups, 6 in each group. The first group was given normal feed (ND), the second group was given high fat diet (HFD), and the second group was used as control. Groups 3-6 were experimental groups, and 2 pmol/g body weight of OCN, ISAP 1 , ISAP 2 , and ISAP 3 were administered daily for 7 weeks, and body weight was monitored once a week. The results of the experiment are shown in Fig. 7A. It is apparent from the figure that a significant weight loss occurred in the ISAP 2 group relative to the HFD control group.
  • ND normal feed
  • HFD high fat diet
  • the feces of the 7th week were collected and baked at 60 ° C for 3 days to ensure drying. Then, 1 mg was taken, soaked in a mixed solution of 1 ml of chloroform and methanol 2 to 1, and then the stool was pulverized by a tissue homogenizer. After taking the supernatant, the triglyceride was detected by Roche blood biochemistry instrument, and the experimental results are shown in Fig. 7B. Compared with the HFD control group, the content of triglyceride in feces of the ISAP 2 group was significantly increased, indicating that intragastric administration of ISAP 2 significantly reduced the absorption of triglycerides in the intestinal tract.
  • mice Six-week-old male wild-type C57BL/6 mice were divided into 6 groups, 6 in each group. The first group was given normal feed (ND), the second group was given high fat diet (HFD), and the second group was used as control. Groups 3-6 were experimental groups, and 2 pmol/g body weight of OCN, ISAP 1 , ISAP 2 , and ISAP 3 were administered daily for 7 weeks, and body weight was monitored once a week. The results of the experiment are shown in Fig. 7A. It is apparent from the figure that a significant weight loss occurred in the ISAP 3 group relative to the HFD control group.
  • ND normal feed
  • HFD high fat diet
  • the feces of the 7th week were collected and baked at 60 ° C for 3 days to ensure drying. Then, 1 mg was taken, soaked in a mixed solution of 1 ml of chloroform and methanol 2 to 1, and then the stool was pulverized by a tissue homogenizer. After taking the supernatant, the triglyceride was detected by Roche blood biochemistry instrument, and the experimental results are shown in Fig. 7B. Compared with the HFD control group, the content of triglyceride in the stool of the ISAP 3 group was significantly increased, indicating that intragastric administration of ISAP 3 significantly reduced the absorption of triglyceride in the intestinal tract.
  • Comparative ISAP 1, ISAP 2, ISAP sequence 3 is found: relative ISAP 1, ISAP 2 has four insertion, two alternative, and a deletion; relative ISAP 1, ISAP 3 has four insertion, 3 substitutions; Compared to ISAP 2 , ISAP 1 has 4 deletions, 2 substitutions, and 1 insertion; in summary, the three polypeptides can be considered as variants, and it is presumed that the three sequences can be used to carry out the technology in the art. Amino acid substitutions, insertions and deletions are well known to persons, provided that their ability to modulate energy metabolism is not significantly reduced, for example by no more than 40%, 30%, 20%, 10%, see Figure 8 for a number of different biological sources. Alignment of homologous sequences, wherein SEQ ID NO.
  • 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.
  • YLDSGLGAPVPYPDPLEP has little difference between the two, when compared, in most cases, the difference is no more than four Substitution of amino acids, therefore, it is presumed that these sequences have similar biological functions and should also be included within the scope of the invention, preferably wherein the total number of deletions, substitutions and insertions does not exceed 4, such as no more than 3, 2, 1. Further, SEQ ID NO. 1 has only one amino acid residue deleted from SEQ ID NO. 18, so it is inferred that SEQ ID NO. 1 also has a function similar to SEQ ID NO.
  • ISAP 1 and ISAP 3 correspond to one insertion at the end of ISAP 2 ; indicating that several amino acid residues can be added at the end of the polypeptide, provided that the ability to regulate energy metabolism is not significantly reduced, for example The reduction is not more than 40%, 30%, 20%, 10%, preferably no more than 5, for example 4, 3, 2, 1 or 0 amino acid residues are added at the end.
  • ISAP 4 features
  • mice of 6 weeks old were divided into 5 groups, 3 rats in each group, and 3 experimental groups were administered with IVP 4 , ISAP 5 and ISAP 6 at 2 pmol/g body weight respectively.
  • Group control daily administration of an equal volume of normal saline by intragastric administration for one week, on the 8th day, 30 minutes after intragastric administration of ISAP 4 , ISAP 5 , ISAP 6 and normal saline, three groups of experimental groups and one control group were intragastrically administered. 200 ⁇ L of sterilized olive oil was applied. One control group was intragastrically administered with 200 ⁇ L of normal saline. After 50 minutes, 95% CO 2 was euthanized and the small intestine was dissected.
  • PSPDPLEP are very similar in sequence, while Species are also very conserved sequences and should therefore have similar biological activities, such that these polypeptides and variants thereof can also be considered to have an effect on the absorption and metabolism of fat by oral administration, i.e., can be known to those skilled in the art. Amino acid substitutions, insertions, and deletions, provided that their ability to modulate energy metabolism is not significantly reduced, for example, by no more than 40%, 30%, 20%, 10%. At the same time, ISAP 4 , ISAP 5 , and ISAP 6 are shorter, resulting in lower cost and better stability, and have great potential for the preparation of drugs for treating diseases associated with abnormal fat metabolism.

Abstract

调节能量代谢的多肽及其在制备用于治疗与能量代谢异常有关之疾病的药物中用途。所述的多肽能够减少脂肪吸收，降低肝脏中的脂肪积累并调节脂肪代谢，并且与一般多肽产品相比具有可以经口施用的优点，因此也可以作为用于调节脂肪代谢的保健品的有效成分。

Description

调节能量代谢的多肽及其用途 技术领域
本发明涉及生物医疗领域，具体地涉及调节能量代谢的多肽及其在制备用于治疗与能量代谢异常，特别是脂肪代谢异常有关之疾病的药物中的用途。
发明背景
肥胖是世界范围内主要且日益增长的健康问题。肥胖也是诱发许多常见病(如动脉粥样硬化、高血压、II型糖尿病、血脂障碍、冠心病、骨关节炎和多种恶性肿瘤)发生的危险因素。它还会通过降低运动能力和生活质量引起更严重的问题。肥胖的发生和由肥胖引起的疾病正在所有的发达国家中增长。
脂肪肝是指肝细胞内脂肪堆积过多导致的病变。脂肪肝的发病原因有很多种，比如酗酒、饮食不合理、久坐等。其在中国的发病率日益增高，成为威胁人们健康的一大威胁。
然而，目前用于治疗肥胖和其引起的疾病，包括非酒精性脂肪肝的药物和方法都具有一定的不足，因此对于肥胖和非酒精性脂肪肝的治疗仍需要开发更有效和低副作用的药物和方法。
骨钙素是由成骨细胞合成和分泌的一种维生素K依赖性钙结合蛋白，一种非胶原酸性糖蛋白，其分子中的维生素K依赖性谷氨酸残基是骨钙素和Ca2+结合的重要功能基团[1,2]。
发明内容
本发明人出人意料的发现了一种来源于骨钙素的能够调节能量代谢的多肽；其具有减少脂肪吸收，降低肝脏脂肪、外周血中血脂、及减小脂肪细胞体积等功能，能够有效清除肝细胞中蓄积的脂肪、降低血脂水平、以及缩小体内脂肪组织，对于治疗脂肪性肝炎、调节高血压及其它心血管疾病起作用。
本发明的一些方面涉及调节能量代谢的多肽，所述多肽由式M1-Za-M2表示，其中：
M1、M2各自独立地为具有不超过5、4、3、2或1个氨基酸残基的多肽区段或者不存在；
Za为Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro，其中：
X1为Tyr、Asn、Asp或不存在，
X2为Gln、Asn、His、Pro、Ser或不存在，
X3为Trp、Gly或不存在，
X4为Leu或不存在，
X5为Pro或Ser，
X6为Ala或Val，
X7为Tyr或Ser，
X8为Thr或Pro，
并且所述Za可任选地具有中氨基酸替换、插入或缺失并且所述氨基酸替换、插入和缺失的总数不超过4，优选不超过3，更优选不超过2，最优选不超过1。
进一步地，本发明的一些实施方案中，所述多肽中的Za选自以下之一：
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。
进一步地，本发明的另一些实施方案中，所述多肽中的M1、M2均不存在；或者在又一些实施方案中，所述多肽中Za为YLYQWLGAPVPYPDPLEP、M1不存在且M2为Arg，即该多肽的氨基酸序列如SEQ ID NO.3所示；在又一些实施方案中，所述多肽中Za为YLGASVPSPDPLEP，M1不存在且M2为Thr，即该多肽的氨基酸序列如SEQ ID NO.18所示。
本发明的另一些方面也涉及调节能量代谢的多肽，其包含选自以下任一序列之至少6个连续氨基酸且其氨基酸残基总数不超过18，例如不超过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。
进一步地，其包含以下任一：
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。
更进一步地，其为
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。
本发明的一些方面涉及本发明的多肽的可药用盐。
本发明的一些方面涉及这样的本发明多肽或其可药用盐，其具有减少脂肪吸收，降低血脂水平，减轻非酒精性脂肪肝、以及缩小体内脂肪组织等作用。
本发明的一些方面涉及多核苷酸，其编码本文上述的多肽。
本发明的一些方面涉及载体，其包含前述的多核苷酸。
本发明的一些方面涉及宿主细胞，其转染有前述的载体并且能够在可表达蛋白质的条件下产生本发明的多肽。
本发明的一些方面涉及药物组合物，其包含治疗有效量的前述本发明多肽或其可药用盐。
在本发明中所述的多肽或其可药用盐或药物组合物具有调节能量代谢，特别是脂肪代谢的作用。
本发明的一些方面涉及本发明的多肽或其可药用盐或药物组合物在制备用于治疗与能量代谢(更优选地脂肪代谢)异常有关之疾病的药物中的用途，所述疾病为可受益于脂肪吸收减少、血脂降低、脂肪消耗增加或脂肪积累减少的疾病，例如肥胖症、II型糖尿病、非酒精性脂肪肝、胰岛素抵抗、高甘油三酯血症、高血糖、高胆固醇、动脉粥样硬化、冠心病。
本发明的一些方面涉及治疗与脂肪代谢异常有关之疾病的方法，其包括向由此需要的对象施用治疗有效量的本发明的多肽或其可药用盐或药物组合物，所述疾病为可受益于脂肪吸收减少、血脂降低、脂肪消耗增加或脂肪积累减少的疾病，例如肥胖症、II型糖尿病、非酒精性脂肪肝、胰岛素抵抗、高甘油三酯血症、高血糖、高胆固醇、动脉粥样硬化、冠心病。
在一些实施方案中，本发明的多肽或其可药用盐或药物组合物可与任何已知用于治疗与脂肪代谢异常有关之疾病的药物和方法组合物一起 施用。
在一些实施方案中，本发明的多肽或其可药用盐或药物组合物用于治疗与脂肪代谢异常有关之疾病，所述疾病为可受益于脂肪吸收减少、血脂降低、脂肪消耗增加或脂肪积累减少的疾病，例如肥胖症、II型糖尿病、非酒精性脂肪肝、胰岛素抵抗、高甘油三酯血症、高胆固醇、动脉粥样硬化、冠心病。
本发明的一些方面涉及本发明的多肽或其可药用盐在制备用于减轻体重之保健品中的用途。
本发明的一些方面涉及用于减轻体重的保健品，其包含本发明的多肽或其可药用盐。
在一些实施方案中，本发明的多肽或其可药用盐或药物组合物可以通过各种常规的方式施用，优选经口施用。
本发明的多肽可以对脂肪的吸收产生直接影响，经口施用减少了常规多肽施用的诸多不便，多肽的残基很少，大大降低了成本，具有作为药物的非常显著的潜在优势。
附图简述
图1示出了使用高脂饲料喂养C57BL/6小鼠12周后获得肥胖和非酒精性脂肪肝模型(Diet-Induced-Obesity&non-alcoholic fatty liver disease，DIO-NAFLD)与正常对照的体重比较；正常饮食(ND)和高脂肪饮食(HFD)。
图2示出了连续6周给高脂肪饮食的DIO-NAFLD小鼠注射不同浓度的ISAP1和OCN(小鼠骨钙素)后，与对照组(仅高脂肪饮食小鼠)相比，注射ISAP1对小鼠的影响。(A)ND对照组、HFD对照组及每日腹腔注射OCN或ISAP1组小鼠附睾脂肪垫的变化；(B)各组中附睾脂肪垫平均质量；(C)各组附睾脂肪垫组织切片的苏木精伊红(H&E)染色；(D)双盲条件下根据H&E染色切片计算获得的平均脂肪细胞表面积。统计学分析：与HFD对照组进行比较。*:P<0.05,**:P<0.01,***:P<0.001。
图3示出了示出了连续6周给高脂肪饮食的DIO-NAFLD小鼠注射不同浓度的ISAP1和OCN后，与对照组(仅高脂肪饮食小鼠)相比，腹腔注射ISAP1对小鼠肝脏的影响。(A)各组小鼠肝脏外观的代表性形象的照片；(B)各组小鼠肝脏组织切片H&E染色；(C)各组小鼠肝脏油红O染色后双盲条件下获得的蓄积非酒精性脂肪肝细胞的表面积。统计学分析：为与HFD对照组进行比较。*:P<0.05,**:P<0.01,***:P<0.001。各组分别有6只小鼠用于分析。
图4示出了连续6周给高脂肪饮食的DIO-NAFLD小鼠注射不同浓度的ISAP1和OCN后，与对照组(仅高脂肪饮食小鼠)相比，注射ISAP1对小鼠肝脏功能和血脂的影响。(A)谷丙转氨酶(ALT)；(B)碱性磷酸酶(ALP)；(C)谷草转氨酶(AST)；(D)血清总胆固醇(TC)；(E)低密度脂蛋白(LDL)；(F)高密度脂蛋白(HDL)。统计学分析：*:与HFD对照组进行比较。*:P<0.05,**:P<0.01,***:P<0.001.#：与ND对照组进行比较。#:P<0.05,##:P<0.01,###P<0.001。各组分别有6只小鼠用于分析。
图5示出了灌胃施用ISAP1和OCN对小肠脂肪吸收的影响。ND饲喂的野生型C57BL/6小鼠灌胃灭菌橄榄油(30分钟)后，腹腔注射ISAP1和OCN后30分钟安乐死小鼠并解剖获得小肠，对近十二指肠段空肠进行冰冻切片并用油红O染色，苏木素复染(A)；双盲条件下对油红O阳性区域通过ImageJ软件进行定量并计算该面积与小肠绒毛总面积的比值(B)。统计学分析：与生理盐水+橄榄油组相比较。***:p<0.001.N＝3。
图6示出了ISAP1和ISAP2分别与人GPRC6A的结合及受体内化。(A)人GPRC6A在Hela细胞中过表达；(B)ISAP1、OCN和OCN-22；以及ISAP2、hOCN和hOCN-22与huGPRC6A过表达的Hela细胞的细胞膜的结合实验；(C)带有Cy-5标记的OCN(Cy5-OCN)、带有Cy-5标记的OCN-22(Cy5-Ocn22)，带有Cy-5标记的ISAP2(Cy5-ISAP2)在GPRC6A过表达的Hela细胞中对GPRC6A内化的影响。
图7示出了在经灌胃施用OCN、ISAP1、ISAP2、ISAP3后七周内对小鼠的影响：(A)小鼠体重变化；(B)小鼠粪便中甘油三酯的含量比较。
图8示出了来自不同物种的ISAP的序列的比较。
图9示出了灌胃施用ISAP4、ISAP5、ISAP6对小肠脂肪吸收的影响。 ND饲喂的野生型C57/b小鼠15只，分为5组，每组3只，三个实验组，分别灌胃施用ISAP4、ISAP5、ISAP6；两组对照，每日灌胃施用生理盐水，持续一周后，三组实验组和一个对照组灌胃施用灭菌橄榄油，一个对照组灌胃施用生理盐水，20分钟后安乐死小鼠并解剖获得小肠，对近十二指肠段空肠进行冰冻切片并用油红O染色，苏木素复染；双盲条件下对油红O阳性区域通过ImageJ软件进行定量并计算该面积与小肠绒毛总面积的比值。统计学分析：与生理盐水+橄榄油组相比较。***:p<0.001.N＝3。
具体实施方式
定义
本文所用的术语“调节能量代谢的多肽”又称作“胰岛素分泌相关多肽(Insulin Secretion Association Peptide，ISAP)”是指来源于骨钙素的或从骨钙素衍生的能够调节能量代谢的多肽及其变体。
本文所使用的术语“保守氨基酸替换”是指用具有相似性质的另一个氨基酸残基对原氨基酸序列进行的替换。例如，赖氨酸残基、精氨酸残基和组氨酸残基在具有碱性侧链方面是相似的。此外，天冬氨酸残基和谷氨酸残基在具有酸性侧链方面是相似的。此外，天冬酰胺残基、谷氨酰胺残基、丝氨酸残基、苏氨酸残基、酪氨酸残基和半胱氨酸残基在具有不带电的极性侧链方面是相似的，并且甘氨酸残基、丙氨酸残基、缬氨酸残基、亮氨酸残基、异亮氨酸残基、脯氨酸残基、色氨酸残基、苯丙氨酸残基和甲硫氨酸残基在具有非极性侧链方面是相似的。此外，酪氨酸残基、苯丙氨酸残基、色氨酸残基和组氨酸残基在具有芳族侧链方面是相似的。因此，对本领域技术人员显而易见的是，在具有上述相似性质的氨基酸组中进行的氨基酸替换不会引起性质的任何变化。
本文中所使用的氨基酸简写及中文名称对照如下
中文名称 三字母简写 单字母简写
丝氨酸 Ser S
苏氨酸 Thr T
天冬酰胺 Asn N
谷氨酰胺 Gln Q
酪氨酸 Tyr Y
半胱氨酸 Cys C
天冬氨酸 Asp D
谷氨酸 Glu E
组氨酸 His H
赖氨酸 Lys K
精氨酸 Arg R
甘氨酸 Gly G
丙氨酸 Ala A
缬氨酸 Val V
亮氨酸 Leu L
异亮氨酸 Ile I
苯丙氨酸 Phe F
甲硫氨酸 Met M
脯氨酸 Pro P
色氨酸 Trp W
本文所使用的术语“与脂肪代谢异常有关之疾病”是指有遗传或环境或二者共同导致的以脂肪代谢紊乱为特征的疾病或其并发症，例如但不限于肥胖症、II型糖尿病、非酒精性脂肪肝、胰岛素抵抗、高甘油三酯血症、高胆固醇、动脉粥样硬化、冠心病。
本文所使用的术语“非酒精性脂肪肝”是指除酒精以外的因素所致的以肝细胞内脂肪过度沉积为主要特征的临床病理综合征。
本文所使用的术语“胰岛素抵抗”是指因用于降低血糖的胰岛素的功能下降而细胞无法有效燃烧葡萄糖的状态。当胰岛素抵抗高时，人体就 生成过多的胰岛素，而导致高血压、异常脂质血症、心脏病和糖尿病等。尤其在II型糖尿病的情况下，由于肌肉和脂肪组织无法识别胰岛素的增加，所以胰岛素不起作用。
本文所使用的未明确定义的术语具有本领域技术人员通常所理解的含义。
具体实施方案
本发明所使用的DMEM购自Sigma中，其中每500ml培养基中补充10％FBS(胎牛血清)，1％非必须氨基酸，1g葡萄糖，0.75g碳酸氢钠，0.1g牛血清白蛋白以及1.5ml HEPES(4-羟乙基哌嗪乙磺酸)。
3T3L1从ATCC购买。
ISAP1序列为Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr(SEQ ID NO.18)。ISAP2的序列为Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.2)。ISAP3的序列为Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala–Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg(SEQ ID NO.3)。ISAP4序列为Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.15)。ISAP5序列为Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.16)。ISAP6序列为Pro-Asp-Pro-Leu-Glu-Pro(SEQ ID NO.14)。
所有的动物实验均通过中国科学院先进技术研究院动物伦理委员会批准，按照伦理委员会的要求进行。
实施例1
ISAP1的功能研究
1、高脂喂养建立肥胖和非酒精性脂肪肝模型
健康6周龄雄性C57BL/6SPF级小鼠购买自广东省实验动物中心，体质量18-22g。分两组，饲养于深圳先进研究院SPF级动物房。对照组：给予小鼠正常饲料(ND)(正常饲料：脂肪，5％；碳水化合物，53％；蛋白，23％；总热量25J/g)，自由摄食和饮水，喂养12周。实验组：给 予小鼠高脂饲料(HFD)(高脂饲料D12451，Research Diets,Inc.)，自由摄食和饮水，喂养12周。检测其生理指标。高脂饲料喂养的小鼠体重超过40g，认为肥胖和非酒精性脂肪肝模型(DIO-NAFLD)构建成功。构建中的小鼠体重与对照组的对比见图1，箭头所指为开始饲喂高脂饲料的时间点。
2、腹腔注射ISAP1对DIO-NAFLD小鼠的影响
2.1腹腔注射ISAP1对附睾脂肪垫的影响
高脂饲料喂养的小鼠体重超过40g，血糖高于10mMol时，对DIO-NAFLD小鼠进行为期6周的ISAP1腹腔注射，每组分别有6只小鼠，将ISAP1溶解于0.01％BSA生理盐水溶液中，每日一次以20pmol/g，2pmol/g剂量腹腔注射到DIO-NAFLD小鼠中，OCN(小鼠骨钙素蛋白)与ISAP1类似，浓度为6pmol/g。6周后，施用95％CO2对小鼠实施安乐死，采集其附睾脂肪垫组织，称重；然后做石蜡切片，通过显微术观察脂肪细胞面积。
实验结果见图2，(A)ND对照组、HFD对照组及每日腹腔注射多肽组小鼠脂肪的整体外观，与HFD对照组相比，施用ISAP1使脂肪组织明显减少；(B)各组小鼠的附睾脂肪垫平均质量，与HFD对照组相比，施用ISAP1使附睾脂肪垫重量显著降低；(C)各组附睾脂肪垫H&E染色，揭示与HFD对照组相比，施用ISAP1使脂肪细胞明显减小；(D)双盲条件下根据H&E染色切片计算获得的平均单个脂肪细胞表面积，说明ISAP1使脂肪细胞明显减小。统计学分析：与HFD对照组进行比较。*:P<0.05,**:P<0.01,***:P<0.001。
2.2腹腔注射ISAP1对肝脏的影响
步骤2.1中，在采集脂肪组织的同时采集肝脏，进行外观拍照，之后取部分肝脏组织进行冰冻切片，使用油红O染色后进行显微镜观察，然后对相同面积进行定量分析，实验结果参见图3。(A)各组小鼠肝脏外观的代表性形象,颜色越淡说明脂肪含量越高；HFD+载剂导致显著的肝脏脂肪积累，腹腔注射ISAP1显著减少了肝脏的脂肪积累(B)各组小鼠肝脏H&E染色，浅色代表细胞内为染色的脂肪；(C)各组小鼠肝脏油红O染色后双盲条件下获得的蓄积非酒精性脂肪肝细胞的表面积，可见在肝脏 中，ISAP1与HFD对照相比，脂肪所占的比例显著降低，表明ISAP1降低了肝脏细胞中的脂肪含量。
2.3腹腔注射ISAP1对小鼠肝脏功能和血脂的影响。
步骤2.1中，在处死小鼠之前，尾静脉采血，使用罗氏血糖仪(型号cobas8000)按照制造商的说明书检测谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、谷草转氨酶(AST)、血清胆固醇，以及低密度脂蛋白(LDL)和高密度脂蛋白(HDL)。实验及结果参见图4，甚至在2pmol/g的剂量下，与HFD对照相比，ISAP1有效降低ALT、ALP、AST、胆固醇以及LDL。
从以上的结果可以看出，ISAP1的腹腔注射可以对小鼠的体内脂肪代谢产生显著影响，减少肝脏细胞和脂肪细胞中的脂肪积累，改善脂肪代谢，有效的缓解非酒精性脂肪肝的进展。
3、ISAP1与人GPRC6A的结合
3.1在Hela细胞中过表达hGPRC6A
1)细胞铺板：将Hela细胞悬液以1.6×105/mL的密度铺板在6孔板中，每孔2mL DMEM培养基，于37℃，5％的CO2中培养24小时。
2)使用Lipofectamine2000(Invitrogen)，按照厂商的说明书将hGPRC6过表达载体(pReceiver-M61)转染到Hela细胞中，转染后4小时更换正常培养基。
3)转染后48小时，弃掉培养基，用无菌PBS清洗板底细胞两次，添加300μL的Trizol溶液，按照RNAiso Plus(TaKaRa)说明书提取细胞RNA，经DNA酶处理后,用SuperScriptT(Invitrogen公司,加拿大)试剂盒经DNAEngine反转录成cDNA。以cDNA为模板,用SYBRGreen法对各时间点的荧光PCR产物进行实时监测分析(Light Cycler Roche公司,德国)，以检测hGPRC6A的表达量。如有需要，使用嘌呤霉素筛选得到稳定表达细胞株，之后使用qPCR检测其基因表达，实验结果如图6A所示，人GPRC6A在Hela中稳定大量表达。
3.2 OCN的不同多肽与过表达hGPRC6A的Hela细胞的细胞膜的结合实验
实验结果见图6B，与OCN相比，ISAP1具有几乎一致的膜结合能力，而OCN-22虽能和GPRC6A结合但亲和性相差了10倍，证明ISAP1是OCN的核心结构域，与受体hGPRC6A相互作用。
4、ISAP1急性灌胃在正常小鼠中对小肠脂肪吸收的影响
使用6周龄雄野生型C57BL/6小鼠，分四组，每组3只，肠道内灌注灭菌橄榄油溶液200μL(Sigma)，30分钟后，腹腔注射OCN和ISAP1(浓度为6pmol/g)，并于30分钟处死小鼠，收集小肠样本。样本为十二指肠到盲肠段，取等长3段。靠近十二指肠段用预冷的生理盐水溶液冲洗后，进行冰冻切片，使用油红O染色观察脂肪吸收。
实验结果参见图5；(A)对近十二指肠段空肠进行冰冻切片并用油红O染色，苏木素复染；(B)双盲条件下对油红O阳性区域通过ImageJ软件进行定量并计算该面积与小肠绒毛总面积的比值。可以看出，相对于盐水对照，ISAP1非常有效的降低了橄榄油吸收，甚至比OCN的效果也明显很多。
5、灌胃施用ISAP1对HFD小鼠的影响
使用6周龄雄野生型C57BL/6小鼠，分6组，每组6只，第1组给予正常饲料(ND)，第2-6组给予高脂饲料(HFD)，第2组作为对照，第3-6组为实验组，每日分别以2pmol/g体重灌胃施用OCN、ISAP1、ISAP2、ISAP3，持续七周，每周监测体重一次。实验结果见图7A，从图中可以明显看出，相对HFD对照组，ISAP1组出现了明显的体重降低。*:P<0.05,**:P<0.01,***:P<0.001。
收集第7周的小鼠粪便，在60℃下烘烤3天，确保烘干，然后取1毫克，用1ml氯仿和甲醇2比1的混合溶液浸泡，然后用组织匀浆仪粉碎粪便，离心后取上清，用罗氏血生化仪检测其甘油三酯，实验结果参见图7B。**:P<0.01,***:P<0.001。与HFD对照组相比，ISAP1组的粪便中甘油三酯的含量明显升高，表明灌胃施用ISAP1显著减少了肠道中甘油三酯的吸收。
实施例2
ISAP2的功能研究
1、ISAP2与人GPRC6A的结合
1.1在Hela细胞中过表达hGPRC6A
1)细胞铺板：将Hela细胞悬液以1.6×105/mL的密度铺板在6孔板中，每孔2mL DMEM培养基，于37℃，5％的CO2中培养24小时。
2)使用Lipofectamine2000(Invitrogen)，按照厂商的说明书将hGPRC6过表达载体(pReceiver-M61)转染到Hela细胞中，转染后4小时更换正常培养基。
3)转染后48小时，弃掉培养基，用无菌PBS清洗板底细胞两次，添加300μL的Trizol溶液，按照RNAiso Plus(TaKaRa)说明书提取细胞RNA，经DNA酶处理后,用SuperScriptT(Invitrogen公司,加拿大)试剂盒经DNAEngine反转录成cDNA。以cDNA为模板,用SYBRGreen法对各时间点的荧光PCR产物进行实时监测分析(Light Cycler Roche公司,德国)，以检测hGPRC6A的表达量。如有需要，使用嘌呤霉素筛选得到稳定表达细胞株，之后使用qPCR检测其基因表达，实验结果如图6A所示，人GPRC6A在Hela中稳定大量表达。
2、ISAP2和hOCN与过表达hGPRC6A的Hela细胞的细胞膜的结合实验
实验方法与实施例中所述基本相同，实验结果见图6B，与hOCN相比，ISAP2具有几乎一致的膜结合能力，而hOCN-22不具备这种能力，结合实施例1中项3.2的结果，证明ISAP1和ISAP2分别是OCN和hOCN的核心结构域，均与受体hGPRC6A相互作用，从而认为ISAP1和ISAP2具有相同的功能，通过hGPRC6A引起后续的信号传导和生物学事件。
2.3带有Cy5标记的OCN、hOCN22，ISAP2在GPRC6A过表达的Hela细胞中促进GPRC6A的内化。
将表达hGPRC6AHela细胞悬液以1.6×105/mL的密度铺板在预先放置明胶涂层盖玻片的24孔板中，每孔0.5mL DMEM，于37℃，5％的CO2中培养24小时。细胞在处理前，用无血清培养基饥饿4h后，每孔添加100nM的Cy5-OCN、Cy5-hOCN22，Cy5-ISAP2，37℃孵育30分钟，使用多聚甲醇固定细胞30分钟，并加入Triton X-100(sigma)孵育10 分钟，DAPI(Sigma)10秒按照厂商的说明书，对细胞和染色，使用荧光共聚焦显微镜观察并拍照，实验结果参见图6C。可见，Cy5-OCN和Cy5-ISAP2在细胞内部有分布，而Cy5-hOCN22则分布在胞外，再次说明了Cy5-OCN和Cy5-ISAP2均可与受体结合并通过内在化作用进入细胞内，从而发挥调节能量代谢的作用。
2.4灌胃施用ISAP2对HFD小鼠的影响
使用6周龄雄野生型C57BL/6小鼠，分6组，每组6只，第1组给予正常饲料(ND)，第2-6组给予高脂饲料(HFD)，第二组作为对照，第3-6组为实验组，分别每日灌胃2pmol/g体重的OCN、ISAP1、ISAP2、ISAP3，持续七周，每周监测体重一次。实验结果见图7A，从图中可以明显看出，相对HFD对照组，ISAP2组出现了明显的体重降低。
收集第7周的小鼠粪便，在60℃下烘烤3天，确保烘干，然后取1毫克，用1ml氯仿和甲醇2比1的混合溶液浸泡，然后用组织匀浆仪粉碎粪便，离心后取上清，用罗氏血生化仪检测其甘油三酯，实验结果参见图7B。与HFD对照组相比，ISAP2组的粪便中甘油三酯的含量明显升高，表明灌胃施用ISAP2显著减少了肠道中甘油三酯的吸收。
实施例3
ISAP3的功能研究
1、灌胃施用ISAP3对HFD小鼠的影响
使用6周龄雄野生型C57BL/6小鼠，分6组，每组6只，第1组给予正常饲料(ND)，第2-6组给予高脂饲料(HFD)，第二组作为对照，第3-6组为实验组，分别每日灌胃2pmol/g体重的OCN、ISAP1、ISAP2、ISAP3，持续七周，每周监测体重一次。实验结果见图7A，从图中可以明显看出，相对HFD对照组，ISAP3组出现了明显的体重降低。
收集第7周的小鼠粪便，在60℃下烘烤3天，确保烘干，然后取1毫克，用1ml氯仿和甲醇2比1的混合溶液浸泡，然后用组织匀浆仪粉碎粪便，离心后取上清，用罗氏血生化仪检测其甘油三酯，实验结果参见图7B。与HFD对照组相比，ISAP3组的粪便中甘油三酯的含量明显升高，表明灌胃施用ISAP3显著减少了肠道中甘油三酯的吸收。
对比ISAP1、ISAP2、ISAP3的序列可知：相对于ISAP1,ISAP2具有4个插入，2个替换，和1个缺失；相对于ISAP1，ISAP3具有4个插入，3个替换；相对于ISAP2，ISAP1具有4个缺失，2个替换，和1个插入；综上所述，可以认为三个多肽互为变体，推定可以以这三个序列为基础，进行本领域技术人员公知的氨基酸替换、插入和缺失，条件是不使其调节能量代谢的能力显著降低，例如降低不超过40％、30％、20％、10％，参见附图8中对多种不同生物来源的同源序列进行比对，其中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之间差异很小，两两比较时，多数情况下差异不超过四个氨基酸的替换，因此可以推定这些序列具有类似的生物学功能，也应当包含在本发明的范围内，优选地，其中缺失、替换和插入的总数不超过4，例如不超过3、2、1。另外SEQ ID NO.1与SEQ ID NO.18相比仅缺失了一个氨基酸残基，因此推断SEQ ID NO.1也具有与SEQ ID NO.18类似的功能。
同时，相对于ISAP2，ISAP1、ISAP3相当于在ISAP2末端具有1个插入；表明可以在多肽的末端添加若干个氨基酸残基，条件是不使其调节能量代谢的能力显著降低，例如降低不超过40％、30％、20％、10％，优选地末端添加不超过5个，例如4个、3个、2个、1个或0个氨基酸残基。
实施例4
ISAP4、ISAP5、ISAP6的功能
ISAP4、ISAP5、ISAP6急性灌胃在正常小鼠中对小肠脂肪吸收的影响
使用6周龄雄野生型C57BL/6小鼠15只，分为5组，每组3只，三个实验组，分别以2pmol/g体重、灌胃施用ISAP4、ISAP5、ISAP6；两组对照，每日灌胃施用等体积生理盐水，持续一周后，第8日，分别灌 胃施用ISAP4、ISAP5、ISAP6以及生理盐水后30分钟，三组实验组和一个对照组灌胃施用灭菌橄榄油200μL，一个对照组灌胃施用生理盐水200μL，50分钟后95％CO2安乐死小鼠并解剖获得小肠，对近十二指肠段空肠进行冰冻切片并用油红O染色，苏木素复染；双盲条件下对油红O阳性区域通过ImageJ软件进行定量并计算该面积与小肠绒毛总面积的比值。统计学分析：与生理盐水+橄榄油组相比较。***:p<0.001.N＝3。。
实验结果参见图9；双盲条件下对油红O阳性区域通过ImageJ软件进行定量并计算该面积与小肠绒毛总面积的比值。可以看出，相对于盐水对照，ISAP4、ISAP5、ISAP6非常有效的降低了橄榄油吸收，证明了他们和ISAP1、ISAP2、ISAP3在生物学功能上相当，同时，参照图8的序列对比，也可以看出SEQ ID NO.12：PVPYPDPLEP与SEQ ID NO.15：SVPSPDPLEP，SEQ ID NO.13：PYPDPLEP与SEQ ID NO.16：PSPDPLEP在序列上相似度非常高，同时在各个物种之间也是非常保守的序列，因而应当具有类似的生物活性，从而可以认为这些多肽及其变体也能够通过口服施用对脂肪的吸收和代谢产生影响，即可以对其进行本领域技术人员公知的氨基酸替换、插入和缺失，条件是不使其调节能量代谢的能力显著降低，例如降低不超过40％、30％、20％、10％。同时，ISAP4、ISAP5、ISAP6更短，因此成本更低，稳定性更好，具有用作制备治疗与脂肪代谢异常有关之疾病的药物的巨大潜力。
本文中提及的所有出版物和专利通过引用整体并入本文，如同每个单独的出版物或专利被具体地且单独地表明通过引用并入。在冲突的情况下，以本申请(包括本文中的任何定义)为准。
等同方案
虽然本文中已经明确地公开了本发明的一些具体实施方案，以上说明书是举例说明性的而非限制性的。对于本领域的技术人员来说，通过浏览本说明书和所附权利要求，本发明的许多变化形式将变得明显。本发明的全部范围应通过参照权利要求、其等同方案的全部范围、以及本说明书和这样的变化来确定。

Claims (19)

  1. 调节能量代谢的多肽，其由式M1-Za-M2表示，其中：
    M1、M2各自独立地为具有不超过5、4、3、2或1个氨基酸残基的多肽区段或者不存在；
    Za为Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro，其中：
    X1为Tyr、Asn、Asp或不存在，
    X2为Gln、Asn、His、Pro、Ser或不存在，
    X3为Trp、Gly或不存在，
    X4为Leu或不存在，
    X5为Pro或Ser，
    X6为Ala或Val，
    X7为Tyr或Ser，
    X8为Thr或Pro，
    并且所述Za可任选地具有氨基酸替换、插入或缺失并且所述氨基酸替换、插入和缺失的总数不超过4。
  2. 根据权利要求1所述的多肽，其中所述Za选自以下之一：
    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。
  3. 根据权利要求2所述的多肽，其中所述M1、M2均不存在。
  4. 根据权利要求2所述的多肽，其中Za为YLYQWLGAPVPYPDPLEP，M1不存在且M2为Arg；或者其中Za为YLGASVPSPDPLEP，M1不存在且M2为Thr。
  5. 调节能量代谢的多肽，其包含权利要求3所述的多肽的至少6个连续氨基酸且其氨基酸残基总数不超过18。
  6. 根据权利要求5所述的多肽，其包含以下之一：
    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。
  7. 根据权利要求6所述的多肽，其为以下之一：
    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。
  8. 权利要求1至7中任一项所述的多肽的可药用盐。
  9. 多核苷酸，其编码权利要求1至7中任一项所述的多肽。
  10. 载体，其包含权利要求9所述的多核苷酸。
  11. 宿主细胞，其转染有权利要求10所述的载体并且能够在可表达蛋白质的条件下产生权利要求1至7中任一项所述的多肽。
  12. 药物组合物，其包含治疗有效量的权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐。
  13. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药 用盐或权利要求12所述的药物组合物在制备用于治疗与能量代谢异常有关之疾病的药物中的用途。
  14. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐或权利要求12所述的药物组合物在制备用于治疗与脂肪代谢异常有关之疾病的药物中的用途。
  15. 权利要求14所述的用途，其中所述疾病为可受益于脂肪吸收减少、血脂降低、脂肪消耗增加或脂肪积累减少的疾病。
  16. 权利要求15所述的用途，其中所述疾病可以为肥胖症、II型糖尿病、非酒精性脂肪肝、胰岛素抵抗、高甘油三酯血症、高胆固醇、动脉粥样硬化、冠心病。
  17. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐或权利要求12所述的药物组合物，其特征在于可经口施用。
  18. 权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐在制备用于减轻体重之保健品中的用途。
  19. 用于减轻体重的保健品，其包含有效量的权利要求1至7中任一项所述的多肽或权利要求8所述的可药用盐。
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