CN115884795A

CN115884795A - Intestinal Probiotics to regulate metabolic adaptation to energy deficit

Info

Publication number: CN115884795A
Application number: CN202180005378.9A
Authority: CN
Inventors: 王一国; 隆艾君; 刘洋
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2023-03-31
Also published as: WO2023000223A1

Abstract

The present disclosure provides: isolating the polypeptide; isolating the nucleic acid; a marker for hyperglycemia; the use of a detection reagent in the preparation of a kit; use of a first agent for the manufacture of a medicament, pharmaceutical composition, food or health product for increasing hunger tolerance; use of a second agent in the manufacture of a medicament, pharmaceutical composition for the treatment or prevention of diabetes; methods for increasing tolerance to starvation; a method for weight loss; methods for treating or preventing diabetes; methods for screening for agents that increase hunger tolerance and methods for screening for agents that treat or prevent diabetes.

Description

Metabolic adaptation of enterosurvivin to regulate energy deficiency

Technical Field

The invention relates to biotechnology, in particular to metabolic adaptation of incretin (Fasmin) to energy deficiency regulation, in particular to isolated polypeptides, isolated nucleic acids, a hyperglycemic marker, the use of a detection reagent in the preparation of a kit, the use of a first reagent in the preparation of a medicament, a pharmaceutical composition, food or health product for improving hunger tolerance, the use of a second reagent in the preparation of a medicament, a pharmaceutical composition for treating or preventing diabetes, a method for improving hunger tolerance, a method for losing weight, a method for treating or preventing diabetes, a method for screening a medicament for improving hunger tolerance and a method for screening a medicament for treating or preventing diabetes.

Background

The intestines are responsible for nutrient absorption and coordinate metabolism in different organs during eating, which is partly a function of gut-derived hormones ^1-3 And (5) controlling. However, it is currently unclear whether the gut plays an important role in metabolism during fasting.

Disclosure of Invention

The object of the present invention is to solve at least one technical problem of the prior art.

The present invention is based on the following findings of the inventors:

here, the inventors found that incretin (a hormone that promotes survival in starvation) is secreted from the intestinal tract and promotes metabolic adaptation to fasting. Mechanistically, incretin is released by the single-pass transmembrane protein Gm11437 during fasting and subsequently binds to the olfactory receptor OLFR796. This incretin-OLFR 796 signaling axis enhances the metabolic response to fasting (including gluconeogenesis, lipolysis and ketone body formation) in mice and promotes fasting-induced hibernation (an adaptive energy-conserving survival strategy) ^4，5 ). The results of the inventors' studies indicate that communication between the gut and other organs via the incretin-OLFR 796 signaling axis is critical for metabolic adaptation to fasting.

Accordingly, the present disclosure provides an isolated polypeptide having the amino acid sequence set forth in SEQ ID NO. 1 or having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO. 1.

MDTILVFSLIIASYDANKKDLRDSSCRLEQLPGIFPKDVRSIRELQMQETHTETKRTTFIQNRTIATLQCLGSDSKVKVNLVYLERRPKVKHILKNLRIIAAPRRNSSASSSCHLIPTSKFQTGSLLKGKAFLPGISQCKVLGASSETFPTTAPSITPGNKEGEKTTSTDTDENLEKR(SEQ ID NO:1)。

According to the embodiment of the present invention, the amino acid sequence shown in SEQ ID NO. 1 is an amino acid sequence of human hormone incretin found by the inventors for the first time. Wherein an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO. 1 is also within the scope of the present application. For example, the amino acid sequence shown in SEQ ID NO. 2 is that of the mouse hormone incretin. 1, which is at least 90% identical to the amino acid sequence shown in SEQ ID NO, and the amino acid sequence of the mouse hormone incretin is also within the scope of the present application.

MDTILVFSLMIASYDSNKNDLRKSSCQVEQWPSFFSEDVRSNKDLVVRVPLEIHTDTKGTPFIQNQPIATLRCLGSGRRVTVHLVYSERRPKVKYIMKNLPVITDLPRNSTASPRCHLRATSQFQNGSLLTAFLPGISQCTVYSAKDRSASSEMVPITTSSTTPRSKGDEATSTGAFPNPLTQGIDMSLKR(SEQ ID NO:2)

Surprisingly, the inventors have found that enterosurvivin is a hormone that promotes survival in energy deprivation. The isolated polypeptides according to embodiments of the invention may confer upon the body the ability to increase tolerance to hunger and to increase survival in energy deficit.

According to one aspect of the present disclosure, there is provided an isolated nucleic acid encoding the polypeptide described above. An isolated nucleic acid according to an embodiment of the present invention encodes an isolated polypeptide as described above. The isolated nucleic acid according to embodiments of the present invention can be used to obtain the above-described isolated polypeptide after introduction into a recipient cell to improve the tolerance of the body to starvation.

According to one aspect of the present disclosure, a marker of hyperglycemia is provided. According to an embodiment of the invention, the marker of hyperglycemia is the polypeptide described above. The inventors found that plasma incretin levels were higher in obese or diabetic models than in healthy models. Therefore, incretins can be used as biomarkers for hyperglycemia and can be used as clinical diagnosis indicators for hyperglycemia.

According to one aspect of the present disclosure, there is provided the use of a detection reagent in the preparation of a kit. According to the embodiment of the invention, the detection reagent is used for detecting the content of the polypeptide in serum, and the kit is used for detecting at least one of the following: blood sugar, plasma beta-hydroxybutyric acid, ketone body, and hepatic acetyl coenzyme A. The inventors found that in Gm11437 gut-specific knockout (IKO) mice, where the incretins were found to be produced by shedding of the extracellular region of Gm11437, blood glucose, plasma β -hydroxybutyrate, ketone bodies and hepatic acetyl coa were significantly reduced in IKO mice, i.e., the incretins levels were closely related to blood glucose, plasma β -hydroxybutyrate, ketone bodies, hepatic acetyl coa. That is, the content of intestinal survivin in vivo can reflect the content of blood sugar, plasma beta-hydroxybutyrate, ketone body and hepatic acetyl coenzyme A in the body. Therefore, the kit prepared by the reagent for detecting the enterosurvivin can be used for detecting blood sugar, plasma beta-hydroxybutyric acid, ketone bodies and hepatic acetyl coenzyme A.

According to one aspect of the present disclosure, there is provided a use of a first agent in the manufacture of a medicament. According to an embodiment of the invention, the first agent is for promoting incretin and the medicament is for at least one of: improving hunger tolerance; reducing insulin sensitivity; increasing blood glucose and/or plasma beta-hydroxybutyrate content; promoting the production of glucose and/or ketone bodies and/or hepatic acetyl-coa; promoting liver fatty acid oxidation; promoting expression of gluconeogenic gene, hibernating related gene, ketogenic gene and/or fatty acid oxidation related gene; promoting the activity of fatty triglyceride lipase. The inventors found that during fasting, incretin is released by the single transmembrane protein Gm11437 and subsequently binds to the olfactory receptor OLFR796. The incretin-OLFR 796 signaling axis enhances the metabolic response of mice to fasting (including gluconeogenesis, lipolysis, and ketone body formation) and promotes fasting-induced hibernation (an adaptive energy-conserving survival strategy). According to the embodiment of the invention, the medicine prepared by using the first agent can be effectively used for improving the hunger tolerance of the body and reducing the insulin sensitivity.

According to one aspect of the present disclosure, there is provided a pharmaceutical composition, food or health product for improving hunger tolerance. According to an embodiment of the invention, the pharmaceutical composition comprises a first agent that promotes incretin. The pharmaceutical composition according to the embodiment of the present invention can be effectively used for improving the hunger tolerance of the body and reducing the insulin sensitivity.

According to one aspect of the present disclosure, there is provided the use of a second agent in the manufacture of a medicament. According to an embodiment of the invention, the second agent is for inhibitingAn incretin, the medicament for use in at least one of: treating or preventing diabetes; increasing insulin sensitivity; reducing blood glucose and/or plasma beta-hydroxybutyrate content; reducing the production of glucose and/or ketone bodies and/or hepatic acetyl-coa; reducing liver fatty acid oxidation; reducing the expression of gluconeogenic genes, hibernating-related genes, ketogenic genes, and/or fatty acid oxidation-related genes; reducing the activity of the fatty triglyceride lipase. The inventors found that plasma incretin levels were significantly increased in obese and diabetic mice fed freely on a diet, while fasting-induced increases in plasma incretin levels were attenuated in obese and diabetic mice. When antibodies neutralized incretin, blood glucose levels were reduced in Wild Type (WT) mice, while the High Fat Diet (HFD) was fed for 16 weeks with an Olfr796 knockout (Olfr 796) ^-/- ) The blood glucose level of the mice was not reduced. According to the embodiment of the present invention, the drug prepared using the second agent can be effectively used for treating or preventing diabetes and increasing insulin sensitivity.

According to one aspect of the present disclosure, there is provided a pharmaceutical composition for treating or preventing diabetes. According to an embodiment of the invention, the pharmaceutical composition comprises a second agent for inhibiting incretin. The pharmaceutical composition according to the embodiment of the present invention can be effectively used for treating or preventing diabetes and increasing insulin sensitivity.

According to one aspect of the present disclosure, a method for increasing tolerance to starvation is provided. According to an embodiment of the invention, the method comprises administering to an individual in need thereof a first agent, wherein the first agent is for promoting incretin.

According to one aspect of the present disclosure, a method of weight loss is provided. According to an embodiment of the invention, the method comprises administering to an individual in need thereof a first agent, wherein the first agent is for promoting incretin.

According to one aspect of the present disclosure, a method for treating or preventing diabetes is provided. According to an embodiment of the invention, the method comprises administering to an individual in need thereof a therapeutically effective amount of a second agent, wherein the second agent is for inhibiting incretin.

According to one aspect of the present disclosure, a method for screening for a drug that increases tolerance to starvation is provided. According to an embodiment of the invention, the method comprises: contacting the drug to be tested with a starvation model; comparing the content of incretin in the starvation model system before and after exposure; wherein an increase in the amount of incretin in the starvation model system after exposure indicates that the drug being screened is the target drug.

According to one aspect of the present disclosure, a method for screening a medicament for treating or preventing diabetes is provided. According to an embodiment of the invention, the method comprises: contacting the screened drug with an obesity or diabetes model; comparing the amount of incretin in the model system for obesity or diabetes before and after exposure; wherein a decrease in the amount of incretin in the model system of obesity or diabetes after exposure indicates that the drug being screened is the target drug.

Further aspects and advantages will be described hereinafter, at least some of which will be apparent from the following description of the accompanying drawings and/or may be apparent to those skilled in the art from the embodiments described below.

Drawings

The above features and advantages and additional features and advantages of the present invention will become more apparent in the following detailed description of the following embodiments when taken in conjunction with the accompanying drawings, in which:

FIG. 1 shows that the incretin is an N-terminal fragment released by Gm11437, wherein, a, is used for siRNA library screening of single transmembrane protein (sTMP) which is positioned on a cytoplasmic membrane and regulates the activity of G6pc-Luc in HepG2 cells. b, correlation between two replicates of the preliminary screen, using a library of sirnas against the 941 sTMP-encoding genes. The results show that G6pc-Luc activity in siRNA knockdown cells is fold-changed (on a log2 scale) relative to cells transfected with non-targeted siRNA. The Spearman's rank correlation coefficient (r = 0.96) between 2 replicates is shown. Knock-down results for AKT1 and INSR (negative regulator, blue), FOXO1 and CRTC2 (positive regulator, green), and CD80, EPHB2, INSRR and C17ORF78 (red) are shown. C, the effect of C17ORF78, C17ORF78/AA (K177A, R178A), incretin (AA 1-178 of C17ORF 78), CD80, EPHB2 or INSRR overexpression on G6pc-Luc activity in HEK293T cells as determined by the transwell method in mouse primary hepatocytes. Data are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-way anova. * P <0.001.NS, no statistical significance. n =6.d, immunoblotting showed expression of C17ORF78, C17ORF78/AA, incretin, CD80, EPHB2 or INSRR in HEK293T cells 48h after transfection. e, alignment of human C17ORF78 and mouse Gm11437. The predicted transmembrane domain (TM) is surrounded by a blue box. The furin (furin) cleavage site in Gm11437 is indicated by red arrows. f, effect of overexpression of Gm11437, gm11437/AA (K190A, R191A) or gut survivin (amino acids 1-191 of Gm 11437) in HEK293T cells analyzed by the Transwell experiment (Transwell assay) on G6pc-Luc activity in mouse primary hepatocytes. Data are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-way anova. * P <0.001.NS, no statistical significance. n =6.g, immunoblot showing expression of Gm11437, gm11437/AA, or incretin in HEK293T cells 48 hours after transfection. h, immunostaining showed the orientation of Gm11437 on the cell membrane of Cos7 cells. Scale bar: 10 μm. i, scheme showing the purification process of the enterosurvivin released by Sf9 cells after Gm11437 cleavage. j, size exclusion chromatography of enterosurvivin representative chromatograms. mAU, slight UV absorption at 280 nm. k, silver staining and immunoblotting showed purified incretins separated by SDS-PAGE. Deglyco, deglycosylation. l, schematic representation shows the in vitro digestion and identification with LC-MS/MS analysis of the enterosurvivin purified from Sf9 cell culture medium. m, protein sequence of enterosurvivin identified by mass spectrometry. n-q, immunoblot showing the glycosylation state of incretins (n), effect of proprotein convertase inhibitor (o) or different proprotein convertase overexpression (p) on Gm11437 cleavage, effect of furin on WT Gm11437 or Gm11437/AA (q) cleavage in HEK293T cells.

FIG. 2 shows the secretion of incretins from the intestine during fasting, where a-b, circadian rhythm (a) and free-feed or fasting time (b) influence plasma incretins levels. ZT is Zeitgeber time. n =5 mice (a), n =6 mice (b). c, qPCR results showed relative mRNA levels of Gm11437 in different tissues of mice. BAT, brown adipose tissue; WAT, white adipose tissue. N =6 mice. d, qPCR results show the relative mRNA levels of C17orf78 in different tissues of humans. N =3 (liver), N =4 (pancreas, intestine, lung, muscle, heart), N =5 (stomach), N =6 (kidney), N =7 (colon). e, generation of Gm11437 intestinal specific knockout (IKO) or Gm11437 liver specific knockout (LKO) mice. f, relative mRNA levels of Gm11437 in WT and IKO mice. g, plasma incretin levels in Wild Type (WT) mice and Gm11437 Intestinal Knockout (IKO) mice that are free-fed or fasted overnight. n =6 mice.

Fig. 3 shows that enterosurvivin promotes mouse survival during fasting, where, a-b, core body temperature (Tb, a) of representative mice during 24-hour fasting, and the number of hibernation occurrences (b) of 24-hour fasted mice. n =7 mice (b). c, autonomic activity of representative mice during 24-hour fasting. And d, fasted mouse survival rate. n =7 mice. Intraperitoneal injection of 400 mug kg after 4 hours of fasting ^-1 Incretin. The gray and white backgrounds (a, c) represent a dark period of 12 hours and a bright period of 12 hours, respectively. Data are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-way analysis of variance (ANOVA, b) or time series test (log-rank test) (d). * P<0.05，**P<0.01，***P<0.001.f, schematically showing the process of purifying incretin-Flag-His from Hi-5 cell culture medium. g, representative chromatogram of enterosurvivin-Flag-His size exclusion chromatography. mAU, slight UV absorption at 280 nm. h, coomassie blue staining (Coomassie staining) showed separate purification of the enterosurvivin-Flag-His by SDS-PAGE. i, intraperitoneal injection (400. Mu.g kg) ^-1 ) Plasma incretin-Flag-His levels later.

Fig. 4 shows that Gm11437 lacks impaired fasting-induced glucose and lipid metabolism, where a-c: blood glucose (a), plasma β -hydroxybutyrate (b) and liver acetyl-coa (c) were fasted overnight for WT mice and Gm11437IKO mice. d, qPCR results show relative mRNA levels of genes involved in lipolysis (Pnlip, pnlip 2, clps, cel), fatty acid oxidation (Cpt 1 a), ketogenesis (Hmgcs 2) and gluconeogenesis (G6 pc, pck 1) in liver extracts from overnight fasted WT and IKO mice. e, immunoblotting showed that Gm11437 lacks effects on hepatic lipolysis and gluconeogenesis. f-h, pyruvate tolerance test (f), glucose tolerance test (g) and insulin resistance test (h) of WT mice and Gm11437IKO mice. i, effect of Gm11437IKO on free-feeding or nocturnal fasting mouse plasma insulin. Data are shown as mean ± s.e.m. n =12 mice (fasted conditions in a, i), n =10 mice (b, c), n =7 mice (d), n =8 mice (f-h), n =6 mice (fed conditions in i). Data are shown as mean ± s.e.m. The comparison of the different groups was performed using Student's t-test. * P <0.01, p <0.001.

FIG. 5 shows that OLFR796 is a receptor for enterosurvivin, in which, a-b, binding experiments of frozen sections of mouse tissue (a) and mouse primary hepatocytes (b). The scale bar of (a) is 20 μm, and the scale bar of (b) is 10 μm. c, coomassie blue staining showed SDS-PAGE separation of GST, GST-incretin, his-ProS2 and His-ProS 2-incretin purified from E.coli. d, determining the binding level of the incretin on the surface of the primary liver cells of the mouse along with the increase of the concentration of the incretin-biotin conjugate. n =3.e, effect of the incretin-Flag-His purified from Hi-5 cells and GST-incretin purified from E.coli on the relative G6pc mRNA levels in mouse primary hepatocytes in the presence or absence of SCH-202676 (SCH, 10M). f, siRNA library screening of GPCRs that modulate GST-incretin binding in HEK293T cells. g, correlation between two replicates of the primary siRNA screen. The results show that the binding of incretin is fold-changed (on a log2 scale) in siRNA knock-out cells compared to non-targeted siRNA transfected cells. Spearman rank correlation coefficient (r = 0.94) between the two replicates is shown. h, GST pull-down experiments (GST pulldown assay) showed the interaction of GST-incretin with OLFR796-Flag or OLFR796-Flag mutants (Mut, R187D, R195D and E197A) in Hi-5 cells. i, schematically showing OLFR796-Flag-His or mutants thereof (R187D, R195D) in Hi-5 cellsAnd E197A). j, OLFR796-Flag-His size exclusion chromatogram representative of chromatography. mAU, slight UV absorption at 280 nm. k, coomassie blue staining showed OLFR796-Flag-His and its mutants (R187D, R195D and E197A) separately purified by SDS-PAGE. l, the binding affinity of incretin to wild type OLFR796 or its mutants (Mut, R187D, R195D and E197A) was quantified by means of Microfuge (MST). Δ F _norm Indicating the change in normalized fluorescence. n =3. Data are expressed as mean ± s.e.m.

FIG. 6 shows the generation and identification of Olfr796 knockout mice, where a, olfr796 is generated by CRISPR-Cas9 ^-/- A mouse. A deletion of 47bp occurred in exon 2 of the Olfr796 gene. b, partial N-terminal sequence of WT-type OLFR796 and Olfr796 ^-/- The complete sequence of the truncated OLFR796 protein product in mice. Shown in red by Olfr796 ^-/- The deletion produced a frameshift of the OLFR796 amino acid in mice. c, PCR analysis showed Olfr796 ^+/+ And Olfr796 ^-/- Mouse generated Olfr796 fragment. d, olfr796 ^+/+ And Olfr796 ^-/- Relative mRNA levels of Olfr796 in mouse liver or skeletal muscle extracts. n =5 mice. e, effect of WT-type OLFR796 or a mutant thereof on binding of incretin in mouse primary hepatocytes, in the presence or absence of incretin. n =3 (e). Data are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-way analysis of variance. * P<0.001.NS, no statistical significance.

FIG. 7 shows that Olfr796 knockout reduces metabolic adaptation to fasting, where, a-c, olfr796 with overnight fasting ^+/+ And Olfr796 ^-/- Blood glucose (a), plasma β -hydroxybutyrate (b) and liver acetyl-coa (c) in mice. d, immunoblotting showed the effect of Olfr796 gene knockout on hepatic lipolysis and gluconeogenesis. e-g, olfr796 ^+/+ And Olfr796 ^-/- Pyruvate tolerance test (e), glucose tolerance test (f) and insulin resistance test (g) in mice. h, effect of Olfr796 gene knockout on plasma insulin levels. i, qPCR results showed that overnight treatment with or without incretin was prohibitedOlfr796 diet ^+/+ And Olfr796 ^-/- Relative mRNA levels of genes involved in lipolysis (Pnlip, pnliprp2, clps, cel), fatty acid oxidation (Cpt 1 a), ketogenesis (Hmgcs 2) and gluconeogenesis (G6 pc, pck 1) in mouse liver extracts. j-l, tb (j) for a representative 24-hour fasted mouse, number of hibernating occurrences (k) and time to hibernate (l) for a mouse during a 24-hour fasted period. m, representative mice that fasted for 24 hours are active autonomously. n, fasted mouse survival. After fasting for 4 hours, 400g kg of the injection is injected into the abdominal cavity ^-1 Incretin. n =8 mice (a-c, e-g, k), n =6 mice (feeding conditions in i, n and h), n =9 mice (fasted conditions in h). Data are shown as mean ± s.e.m. Comparisons of the different groups were performed using student's t-test (a-c, e-g), two-way analysis of variance (h, k, l), or time series test (n). * P<0.05，**P<0.01，***P<0.001.NS, no statistical significance.

FIG. 8 shows the correlation of incretins and diabetes, where a, free-fed or nocturnal fasting lean mice, db/db and ob/ob mice plasma incretins levels. b, plasma incretin levels in mice fed either free-fed or nocturnally fasted Regular Diet (RD) or High Fat Diet (HFD). Normal humans or type 2 diabetic patients are fasted or fed for 2 hours (postprandial) at night for plasma incretin levels. d, anti-enterosurvivin antibody (200 g kg) ^-1 ) Olfr796 for RD or HFD feeding ^+/+ And Olfr796 ^-/- Effect of blood glucose in mice. Data are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-way analysis of variance (a-d). * p is a radical of<0.05，***p<0.001.NS, no statistical significance. n =6 mice (a, b), n =15 humans (c), n =5 mice (d).

Fig. 9 shows that enterosurvivin-OLFR 796 enhances the metabolic response of mice to fasting, wherein enterosurvivin is secreted from the intestine during fasting and binds to OLFR796, thereby promoting fasting-induced hibernation and metabolism including gluconeogenesis, fat breakdown, fatty acid oxidation, and ketone body production. Thus, the incretin-OLFR 796 signaling axis enhances the survival response of mice to energy deficit.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The above features and advantages and additional features and advantages of the present invention will become more apparent in the light of the following detailed description of exemplary embodiments thereof, as illustrated in the accompanying drawing.

The embodiments described herein with reference to the drawings are illustrative and explanatory and are intended to be understood as the invention as a whole. The examples should not be construed as limiting the scope of the invention. Throughout the specification, the same or similar elements and elements having the same or similar functions are denoted by the same reference numerals.

Isolated polypeptides and isolated nucleic acids

According to one aspect of the disclosure, the disclosure provides an isolated polypeptide having the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID No. 1. According to the embodiment of the invention, the amino acid sequence shown in SEQ ID NO. 1 is the amino acid sequence of human hormone incretin discovered for the first time by the inventor. Amino acid sequences which have at least 90% identity (e.g.90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) to the amino acid sequence shown in SEQ ID NO. 1 are also included in the scope of the present application. For example, the amino acid sequence shown in SEQ ID NO. 2 is that of the mouse hormone incretin. 1, which is at least 90% identical to the amino acid sequence shown in SEQ ID NO, and the amino acid sequence of the mouse hormone incretin is also within the scope of the present application. The inventors have surprisingly found that the hormone incretin promotes survival in energy deficit. Isolated polypeptides according to embodiments of the invention can confer increased tolerance to starvation and increased ability to survive energy deficit.

In one aspect of the invention, there is provided an isolated nucleic acid encoding the polypeptide described above. An isolated nucleic acid according to an embodiment of the invention encodes an isolated polypeptide as described above. The isolated nucleic acids according to embodiments of the invention can be used to obtain the above-described isolated polypeptides after introduction into recipient cells to improve the tolerance of the body to starvation.

According to embodiments of the disclosure, the isolated nucleic acid has the sequence shown in SEQ ID NO 3 or SEQ ID NO 4, or a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to the nucleotide sequence shown in SEQ ID NO 3 or SEQ ID NO 4.

ATGGATACCATCTTAGTCTTCAGCCTAATGATTGCATCCTATGATTCCAACAAGAACGACCTCAGAAAGAGCAGCTGCCAAGTGGAACAGTGGCCCAGCTTCTTCTCAGAGGATGTGAGGAGCAACAAGGATTTGGTAGTAAGAGTTCCTCTAGAAATTCACACAGATACCAAAGGGACCCCATTCATCCAAAATCAGCCGATAGCTACCCTGCGGTGTCTTGGCTCTGGAAGGAGAGTGACAGTCCACCTTGTATATTCAGAGAGAAGGCCAAAGGTCAAGTACATTATGAAGAACCTGCCAGTCATTACTGATCTTCCTAGAAACAGCACTGCTTCCCCAAGATGTCACCTCAGAGCCACATCTCAGTTTCAGAATGGATCCCTTCTAACAGCTTTTTTACCAGGGATCTCACAATGCACGGTCTACTCAGCTAAGGACAGATCTGCTTCATCAGAGATGGTGCCCATCACTACCTCTTCCACAACTCCTAGAAGTAAAGGAGATGAAGCCACAAGCACTGGGGCTTTTCCCAACCCTTTAACACAAGGCATAGACATGTCCTTAAAGAGGTGA(SEQ ID NO:3)

ATGGATACCATCTTGGTCTTCAGCCTAATCATTGCATCCTATGATGCCAACAAGAAAGACCTCAGAGATAGCAGTTGCCGACTGGAACAGCTGCCTGGGATCTTCCCAAAAGACGTGAGAAGCATCAGAGAATTGCAAATGCAAGAAACTCACACAGAAACCAAAAGGACAACATTCATTCAAAACCGGACTATAGCTACCCTGCAGTGCCTTGGCTCTGACAGCAAAGTAAAAGTCAACCTTGTATATTTGGAGAGAAGGCCAAAGGTCAAGCATATTTTGAAGAACCTGAGAATCATTGCTGCTCCCCGCAGAAACAGCTCTGCCTCCTCAAGCTGTCACCTAATCCCCACATCCAAGTTTCAGACTGGATCTCTTCTAAAAGGCAAAGCTTTTTTACCAGGGATCTCACAATGTAAAGTCCTGGGGGCTTCATCAGAGACTTTTCCCACCACTGCCCCTTCTATAACTCCTGGGAATAAAGAAGGAGAGAAAACTACAAGTACCGACACAGATGAGAACCTAGAGAAGAGATGA(SEQ ID NO:4)

For comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [ the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding position in the second sequence ] by [ the total number of nucleotides/amino acids in the first sequence ] multiplied by [100% ], wherein each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence is considered a difference of a single nucleotide (position) compared to the first nucleotide sequence.

Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms (e.g., NCBI Blast v 2.0) using standard settings for sequence alignment.

Other techniques, computer algorithms and arrangements for determining the degree of sequence identity are described, for example, in WO 04/037999, EP 0 967, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.

For comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [ the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residue at the corresponding position in the second amino acid sequence ] by [ the total number of nucleotides in the first amino acid sequence ] multiplied by [100% ], wherein each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence is considered a difference of a single amino acid residue (position) as compared to the first amino acid sequence, as defined herein as an "amino acid difference".

Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as the algorithms mentioned above for determining the degree of sequence identity of nucleotide sequences, again using standard settings.

In general, to determine the percentage of "sequence identity" between two amino acid sequences according to the above calculation method, the amino acid sequence with the largest number of amino acid residues will be referred to as the "first" amino acid sequence, and the other amino acid sequence will be referred to as the "second" amino acid sequence.

In addition, in determining sequence identity between two amino acid sequences, one skilled in the art may consider so-called "conservative" amino acid substitutions, which may be generally described as amino acid substitutions, i.e., the substitution of one amino acid residue for another with a similar chemical structure and having little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; such alternative (preferred) types and/or combinations may be selected in accordance with the relevant teachings of WO 04/037999 and WO 98/49185, and from other references cited therein.

Such conservative substitutions are preferably those in which one amino acid in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: alanine, serine, threonine, proline, and glycine; (b) Polar, negatively charged residues and their (uncharged) amides: aspartic acid, asparagine, glutamic acid and glutamine; (c) polar, positively charged residues: histidine, arginine and lysine; (d) large aliphatic, nonpolar residues: methionine, leucine, isoleucine, valine, and cysteine; (e) aromatic residues: phenylalanine, tyrosine and tryptophan.

Particularly preferred conservative substitutions are as follows: alanine is replaced by glycine or serine; arginine was replaced with lysine; asparagine is replaced with glutamine or histidine; asparagine was replaced with glutamic acid; cysteine to serine; glutamine was replaced with asparagine; glutamic acid to aspartic acid; glycine to alanine or proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to isoleucine or valine; lysine is replaced by arginine, glutamine or glutamic acid; methionine to leucine, tyrosine or isoleucine; phenylalanine to methionine, leucine, or tyrosine; serine to threonine; threonine is replaced by serine; tryptophan to tyrosine; tyrosine to tryptophan; and/or phenylalanine to valine, isoleucine or leucine.

Any amino acid substitution of the polypeptides described herein may also be based on analysis of the frequency of amino acid mutations between homologous proteins of different species developed by Schulz et al (Schulz et al, principles of Protein Structure, springer-Verlag, 1978), on analysis of the Structure-forming potential developed by Chou and Fasman (Biochemistry 13, 1974 and adv. Enzymol., 47-149, 1978) and on analysis of the pattern of hydrophobicity in proteins developed by Eisenberg et al(Eisenberg et al.,Proc.Nat.Acad Sci.USA 81:140-144,1984；Kyte&Doolittle, J mol.biol.157:105-132,198 1 and Goldman et al, ann.Rev.Biophys.chem.15:321-353, 1986), which are all incorporated herein by reference. Information regarding the primary, secondary, and tertiary structure of the nanobodies is provided in the description herein and in the background art cited above. Also, to achieve this goal, the crystal structure of the VHH domain of llama (llama) is provided, for example, in the following literature: desmyter et al, nature Structural Biology, vol.3,9,803 (1996); spinelli et al, natural Structural Biology (1996); vol.3,752-757; and Decanniere et al, structure, vol.7,4,361 (1999). Further information provides amino acid residues which are at the classical V _H Formation of V in structural domains _H /V _L Interfaces and potential camelizing subsistitions at these locations.

An amino acid sequence and a nucleic acid sequence are "identical" if they share 100% sequence identity (as defined herein) over their entire length.

A nucleic acid sequence or amino acid sequence is considered to be "(in) substantially isolated (form)", e.g. when it has been separated from at least one other component associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component, as compared to its native biological source and/or the reaction medium or culture medium from which it was obtained. In particular, a nucleic acid sequence or amino acid sequence is considered "substantially isolated" when it is purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, up to more than 1000-fold. The nucleic acid sequence or amino acid sequence "in substantially isolated form" is preferably substantially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, e.g. polyacrylamide gel electrophoresis.

At least one amino acid site of the polypeptide is glycosylated.

According to an embodiment of the invention, at least one amino acid site of the polypeptide is glycosylated. The inventors found that the enterosurvivin secreted upon starvation was highly glycosylated.

Markers for hyperglycemia

According to one aspect of the present disclosure, a hyperglycemic marker is provided. According to an embodiment of the invention, the marker of hyperglycemia is the polypeptide described above. The inventors found that plasma incretin levels were higher in obese or diabetic models than in healthy models. Therefore, incretins can be used as biomarkers for hyperglycemia and can be used as clinical diagnosis indicators for hyperglycemia.

Application of detection reagent in preparation of kit

According to one aspect of the present disclosure, there is provided the use of a detection reagent in the preparation of a kit. According to the embodiment of the disclosure, the detection reagent is used for detecting the content of the polypeptide in serum, and the kit is used for detecting at least one of the following: blood sugar, plasma beta-hydroxybutyric acid, ketone body and hepatic acetyl coenzyme A. The inventors found that in Gm11437IKO mice (which the inventors found that enterosurvivin is produced by the shedding of the extracellular region of Gm 11437), blood glucose, plasma β -hydroxybutyrate, ketone bodies and hepatic acetyl coa were all significantly reduced in IKO mice, i.e. the incretin levels were closely related to blood glucose, plasma β -hydroxybutyrate, ketone bodies, hepatic acetyl coa levels. That is, the content of intestinal survivin in vivo can reflect the contents of blood sugar, plasma beta-hydroxybutyric acid, ketone body and hepatic acetyl coenzyme A of the organism. Therefore, the kit prepared by the incretin detection reagent can be used for detecting blood sugar, plasma beta-hydroxybutyric acid, ketone body and hepatic acetyl coenzyme A.

Use of a first agent in the manufacture of a medicament

In one aspect of the invention, there is provided the use of a first agent in the manufacture of a medicament. According to an embodiment of the disclosure, the first agent is for promoting incretin, the medicament is for at least one of: improving hunger tolerance; reducing insulin sensitivity; increasing blood glucose and/or plasma beta-hydroxybutyrate content; promoting the production of glucose and/or ketone bodies and/or hepatic acetyl-coa; promoting liver fatty acid oxidation; promoting expression of gluconeogenic genes, hibernating-related genes, ketogenic genes and/or genes involved in fatty acid oxidation; promoting the activity of fatty triglyceride lipase. The inventors found that during fasting, incretin is released by the single transmembrane protein Gm11437 and subsequently binds to the olfactory receptor OLFR796. The incretin-OLFR 796 signaling axis enhances the metabolic response of mice to fasting (including gluconeogenesis, lipolysis, and ketone body formation) and promotes fasting-induced hibernation, an adaptive energy conservation survival strategy. According to the embodiment of the invention, the medicine prepared by using the first agent can be effectively used for improving the hunger tolerance of the body and reducing the insulin sensitivity.

It should be noted that the "incretin" mentioned in the present application includes all possible methods of improving the in vivo function of incretins, such as increasing the amount of incretins, increasing the expression or secretion of incretins, enhancing the activity of incretins, enhancing the combination of incretins with OLFR796, increasing the expression of OLFR796 or enhancing the activity of OLFR796.

Thus, for example, the first reagent comprises at least one selected from: an incretin or functional fragment of an incretin; an agent for overexpression of an incretin or an incretin functional fragment; an agent for promoting shedding of an incretin from Gm 11437; an enterosurvivin activator; an agent that enhances binding of incretin to OLFR 796; an agent that overexpresses OLFR 796; OLFR796 activator. Wherein an incretin functional fragment refers to a functional fragment or a regional fragment capable of independently performing an incretin function. Wherein the amount of incretin can be increased using an incretin or functional fragment of an incretin and an agent that overexpresses the incretin or functional fragment of an incretin. The secretion of the incretin is promoted by an agent for promoting the shedding of the incretin from Gm11437. The use of an incretin activator enhances the activity of incretin. An agent that enhances the binding of incretin to OLFR796 can be used to promote the binding of incretin to OLFR796. Agents for over-expressing OLFR796 may be used to increase the amount of OLFR796, and OLFR796 activators may also be used to enhance the activity of OLFR796.

According to an embodiment of the invention, the improvement of hunger tolerance is achieved by: promoting gluconeogenesis, lipolysis and/or ketone body production; promoting fasting-induced hibernation; adaptive energy conserving survival strategies such as hibernation, lowering core body temperature (Tb) and/or reducing voluntary activity and/or increasing energy mobilization.

According to embodiments of the disclosure, the gluconeogenic genes are G6pc and Pck1, the hibernation-related genes are Pnlip, pnlip 2, clps and Cel, the ketogenic gene is Hmgcs2, and/or the gene involved in fatty acid oxidation is Cpt1a.

According to an embodiment of the present invention, the agent that promotes the shedding of incretins from Gm11437 is furin.

Use of a second agent in the manufacture of a medicament

According to one aspect of the present disclosure, there is provided the use of a second agent in the manufacture of a medicament. According to embodiments of the present disclosure, the second agent is for inhibiting incretin, and the medicament is for at least one of: treating or preventing diabetes; increase insulin sensitivity; reducing blood glucose and/or plasma beta-hydroxybutyrate content; reducing the production of glucose and/or ketone bodies and/or hepatic acetyl-coa; reducing liver fatty acid oxidation; reducing the expression of gluconeogenic genes, hibernating-related genes, ketogenic genes, and/or genes involved in fatty acid oxidation; reducing the activity of the fatty triglyceride lipase. The inventors found that plasma incretin levels were significantly increased in obese and diabetic mice fed freely on a diet, whereas fasting-induced increases in plasma incretin levels were attenuated in obese and diabetic mice. When antibodies neutralized enterosurvivin, the blood glucose levels of WT mice were reduced, while feeding HFD for 16 weeks of Olfr796 ^-/- The blood glucose level of the mice was not reduced. According to the embodiment of the present invention, the medicine prepared using the second agent can be effectively used for treating or preventing diabetes and increasing insulin sensitivity.

It is noted that reference to "inhibiting incretins" in this application includes all methods that may inhibit the function of incretins in vivo, such as reducing the amount of incretins, including neutralizing the incretins, reducing the expression or secretion of incretins, reducing the activity of incretins, reducing the binding of incretins to OLFR796, reducing the expression of OLFR796 or reducing the activity of OLFR796.

Thus, for example, the second reagent comprises at least one selected from: an agent for neutralizing an incretin or an incretin functional fragment; an agent for silencing an incretin or functional fragment of an incretin; an agent for inhibiting the shedding of incretin from Gm 11437; an incretin inhibitor; an agent for inhibiting binding of incretin to OLFR 796; an agent for silencing OLFR 796; OLFR796 inhibitors; OLFR796 mutagen. Wherein the incretin can be reduced using an agent that neutralizes and silences an agent for the incretin or functional fragment of the incretin. An agent that inhibits the shedding of incretins from Gm11437 can be used to reduce the secretion of incretins. An incretin inhibitor can be used to reduce the activity of incretin. Agents that inhibit the binding of incretin to OLFR796 can be used to reduce the binding of incretin to OLFR796. An agent that silences OLFR796 can be used to reduce the receptor OLFR796. OLFR796 inhibitors may be used to inhibit the activity of OLFR796. OLFR796 can be mutated using an OLFR796 mutagen, whereby OLFR796 loses its binding activity to incretin.

According to embodiments of the disclosure, the agent for neutralizing an incretin or functional fragment of an incretin is an antibody that specifically binds to an incretin or functional fragment of an incretin. Methods of constructing antibodies are known in the art, for example, newcomb C, newcomb AR.antibody production, polyclonal-derived biphenotherapeutics.J.Chromatogr B analytical Technol Biomed Life Sci.2007 Mar 15;848 (1) 2-7. Doi; 53 (3) 111-7.doi; PMCID 1186915. Unless otherwise indicated, all methods, steps, techniques and operations not described in particular detail may be performed and have been performed in a manner apparent to those of ordinary skill in the art.

According to embodiments of the present disclosure, a mutagen in OLFR796 causes at least one of the following mutations in OLFR 796: R187D, R195D and E197A.

Pharmaceutical composition, food or health product

According to one aspect of the present disclosure, there is provided a pharmaceutical composition, food or health product for improving tolerance. According to an embodiment of the present disclosure, the pharmaceutical composition, food or health product includes a first agent for promoting incretin. The pharmaceutical composition, food or health product according to the embodiment of the present invention can be effectively used for improving the hunger tolerance of the body and reducing the insulin sensitivity.

According to one aspect of the present disclosure, a pharmaceutical composition for treating or preventing diabetes is provided. According to an embodiment of the invention, the pharmaceutical composition comprises a second agent for inhibiting enterosurvivin. The pharmaceutical composition according to the embodiment of the present invention can be effectively used for treating or preventing diabetes and increasing insulin sensitivity.

The pharmaceutical compositions disclosed herein may be used by any of the following routes: oral, aerosol inhalation, topical, rectal, nasal, vaginal, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrapulmonary, intrathecal, intracerebroventricular, intrasternal, or intracranial injection or infusion, or by explant. The intraperitoneal injection administration route is preferred.

The pharmaceutical compositions of the present invention may be administered in unit dosage forms. The dosage form may be in liquid form or in solid form. Liquid forms include true solutions, colloids, microparticles, suspensions. Other dosage forms include tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, implant, patch, liniment, etc.

The pharmaceutical composition for treating or preventing diabetes provided by the invention also comprises other medicines for treating or preventing diabetes.

The pharmaceutical composition for improving hunger tolerance provided by the invention also comprises other medicines for inhibiting hunger.

Methods for increasing hunger tolerance or weight loss

According to one aspect of the present disclosure, a method of increasing tolerance to starvation is provided. According to an embodiment of the invention, the method comprises administering to an individual in need thereof a first agent, wherein the first agent is for promoting incretin.

According to embodiments of the disclosure, the individual is administered the first agent after experiencing at least 18 hours of hunger. The inventors found that plasma incretin levels are elevated after fasting. The maximum level (700 pM) is reached after 18 hours of fasting and decreases after continued fasting. Administration of the first agent to the individual after experiencing at least 18 hours of hunger is effective to prolong hunger tolerance. The longer the fasting effect can be, the better the weight loss effect.

Method for treating or preventing diabetes

According to one aspect of the present disclosure, a method of treating or preventing diabetes is provided. According to an embodiment of the invention, the method comprises administering to an individual in need thereof a therapeutically effective amount of a second agent, wherein the second agent is for inhibiting incretin.

According to embodiments of the present disclosure, administration is achieved by intraperitoneal injection.

Method for screening drugs for improving hunger tolerance

In one aspect of the invention, a method for screening for an agent that increases tolerance to starvation is provided. According to an embodiment of the present disclosure, the method includes: contacting the drug to be tested with a starvation model; comparing the amount of incretin in the starvation model system before and after the contacting, wherein an increase in the amount of incretin in the starvation model system after the contacting indicates that the screening drug is the target drug.

According to an embodiment of the present invention, the starvation model is a starvation-treated intestinal epithelial cell or mouse model.

According to an embodiment of the present invention, the hunger model is a mouse model subjected to hunger treatment, and the contact is achieved by administering the test drug to the hunger model by intraperitoneal injection.

Method for screening drug for treating or preventing diabetes

According to one aspect of the present disclosure, a method for screening a medicament for treating or preventing diabetes is provided. According to an embodiment of the invention, the method comprises: contacting the screening drug with an obesity or diabetes model; comparing the content of incretin in the obesity or diabetes model system before and after the exposure; wherein a decrease in the content of incretin in the post-exposure obesity or diabetes model system indicates that the screened drug is the target drug.

According to an embodiment of the invention, the obesity or diabetes model is a mouse model induced by a high fat diet.

Examples of the invention

Method

Human research

Blood samples from healthy persons and type 2 diabetic patients were centrifuged at 4 ℃ immediately after collection, then separated and stored at-80 ℃. All participants provided written informed consent. The study was approved by the institutional review board of Qinghua university and the institutional review board of the second subsidiary hospital of Zhejiang university of traditional Chinese medicine. Blood glucose was measured using AU5000 (Beckman Coulter). HbA1c was detected using Cobas 8000 (Roche) and plasma insulin was detected using Variant II Turbo (Bio-Rad). Serum incretin levels were detected by sandwich ELISA.

Mouse strains and experiments

Mice were placed in a temperature controlled environment using a 12 hour light/12 hour dark cycle (light was turned on at 7 am as ZT0, light was turned off at 7 pm as ZT 12), and the ambient temperature was controlled at about 21 ℃ to allow free access to food and water. Animals can freely take water at any time, and food can be removed only when needed in experiments. In the high fat diet feeding experiment, the regular diet (D12450J, study diet) was replaced by a diet containing 60 kcal% fat (D12492, study diet). In the antibody neutralization assay, an in vitro assay was performed to test the potential of rat IgG antibodies to neutralize incretin. Abdominal injection of anti-enterosurvivin antibody (200. Mu.g kg) into mouse ^-1 ) Or isotype matched immunoglobulin G (IgG). Animal feeding was in accordance with all relevant ethical regulations for animal testing and studies. All animal experiments were approved by the university of Qinghua animal protection and use Committee.

B6/JGpt-Lep ^em1Cd25 /Gpt (T001461) and BKS-Lepr ^em2Cd479 the/Gpt (T002407) mice were purchased from GemPharmatech. Mice carrying the floxed allele of Gm11437 were obtained from viewsolidd Biotech. To generate liver-specific or gut-specific Gm11437 knockout mice, gm11437 was generated ^fl/fl Mice were crossed with mice expressing Cre recombinase transgenes from either the liver-specific albumin promoter (Alb-Cre) or the gut-specific villin 1 promoter (Vil 1-Cre) to delete exon 1. PCR typing was performed on Gm11437 knockout mice using primers that detect the following: 3' LoxP site of target Gm11437 allele: forward, 5 'GTTCTCATTGTTGGCATCAT-3'; reverse, 5 'AGCCTACTGTCTCATCTGT-3'. Cre: forward, 5 'GCCTGCATTACCGTCGATGC-3'; cre reverse, 5 'CAGGGTGTTATAAGCAATCCC-3'. Internal reference of Cre: forward, 5 'CTAGGCCACAGAATTGAAAGATCT-3'; reverse, 5 'sand glass ceramic powder GTAGGTGGAAATTCTAGCATCCK 3'. By injecting Cas9 mRNA (100 ng. Mu.l) into fertilized eggs ^-1 ) And 2 sgRNAs (50 ng. Mu.l each) ^-1 ) Of Olfr796. High quality cleavage embryos from 2-cell stage to blastocyst stage were transferred to the oviduct of the matched recipient mouse. Heterozygote mice expressing Olfr796 deleted were crossed to each other to generate knockout mice. PCR was performed using primers 5-. All mice were maintained on a C57BL/6J background.

Thermography and fasting induced hibernation

Mice were housed individually and implanted with a telemetric temperature and activity probe (G2E-MITTER, starr Life Science) in the abdomen. After at least 5 days of recovery, mice were recorded in standard cages placed on a radio frequency receiving platform (ER 4000, star Life Science). Gross movement and core body temperature were measured using vitaview telemetry data acquisition software (star Life Sciences). After baseline measurements (free food and water intake), mice were placed in new cages, and food was removed at ZT12 to induce hibernation. Initial hibernation development was observed after fasting for about 7 hours. The death of the mice was defined as: core body temperature (Tb) <28 ℃, then falls rapidly below ambient temperature.

Metabolic Studies

Blood glucose levels were measured using a LifeScan autoglucometer. Abdominal injection of glucose (1 g.kg) after overnight fasting ^-1 ) Glucose Tolerance Tests (GTT) were performed. Human regular insulin (1 u kg) was injected intraperitoneally 5 hours after fasting ^-1 ) An insulin resistance test was performed. In the pyruvate tolerance experiment, mice were fasted overnight and were injected intraperitoneally with pyruvic acid (1 g kg) ^-1 ). Liver acetyl coenzyme A (MAK 039, sigma), plasma insulin (10-1247-01, mercodia) and plasma hydroxybutyric acid (K632, bioVision) were measured according to the manufacturer's instructions.

Sandwich ELISA

Sandwich enzyme-Linked immunosorbent assay (ELISA) As described previously ²⁷ Is carried out. For the mouse incretin sandwich ELISA, a rat monoclonal anti-incretin antibody (1. For human enterosurvivin sandwich ELISA, a mouse polyclonal anti-enterosurvivin antibody (1. Anti-rabbit secondary antibodies conjugated with HRP (1706515, bio-Rad) were used to generate signals. The rat anti-incretin monoclonal antibody and the rabbit anti-incretin polyclonal antibody are produced by Beijing Prorevo Biotechnology Ltd. Mouse anti-incretin polyclonal antibodies were prepared in the animal laboratory at the university of Qinghua.

Cell culture

HEK293T, cos7 and HepG2 (ATCC) cells containing 10% FBS (HyClone) and 1%00mg ml ^-1 Penicillin-streptomycin DMEM maintained at 37 ℃ and 5% CO ₂ In (1). Hi-5 and Sf9 cells were cultured in SIM HF medium (Single Biological) and SIM SF medium (Single Biological), respectively, at a temperature of 27 ℃. Mouse Primary hepatocytes the methods described previously ^27、39 Separate, culture in M199 medium containing 2% FBS and 0.2% bsa until adherence, then culture was continued in M199 medium without FBS.

Reagent

Glucagon (HY-P0082, 100nM, medChemexpress), proprotein convertase inhibitor 1 (537076, 5. Mu.M, calbiochem), proprotein convertase inhibitor 2 (ALX-260-022, 5. Mu.M, enzo), SCH-202676 (1400, 10. Mu.M, tocris), U73122 (S8011, 5. Mu.M, serleck), YM-254890 (257-00631, 2. Mu.M, wako) and Xestospongin C (64950, 2. Mu.M, cayman Chemical) were used in this study.

Plasmids

Myc-FLAG labeled CD80 (MR 227446), EPHB2 (MR 225676), INSRR (MR 217196), PCSK2 (MR 226625), PCSK6 (MR 215393), PCSK7 (MR 210558), and GNAQ (MR 205488.) were all purchased from OriGene Technologies, inc. V5-labeled PCSK1, furin and PCSK5 were purchased from GE healthcare. Olfr796 was synthesized by TsingKe Biological Technology, inc. C17ORF78 and Gm11437 were amplified from human and mouse liver cDNA libraries, respectively. G6pc-Luc has been described previously ⁴⁰ . All expression constructs used in this study were confirmed by sequencing.

siRNA screening

To determine which single transmembrane proteins (sTMP) ON the cell membrane affected G6pc-Luc, hepG2 cells stably expressing G6pc-Luc were transfected with siRNA encoding RSV-Luc and a human sTMP selected from an ON-TARGET + whole genome siRNA library (Dharmacon). 48 hours after siRNA addition, luciferase assays were performed using the dual luciferase reporter assay system (E1980, promega) and normalized with co-transfected RSV-Luc activity. To determine which GPCR is a receptor for incretin, HEK293T cells were transfected with sirnas for human GPCR-encoding genes selected from an ON-TARGET + whole genome siRNA library (Dharmacon). 48 hours after siRNA addition, cells were incubated with GST-incretin (100 nM) for 30 minutes. Cells were stained with anti-GST (WH 117206, abcronal) antibody and analyzed with FACS Calibur flow cytometer (BD Biosciences).

Protein expression and purification

To purify the cleaved product of Gm11437 from the culture medium, gm11437 was first expressed in Sf9 insect cells using the pFastBac baculovirus system (Invitrogen). Conditioned medium was collected 48h after viral infection, diluted 2-fold with buffer I (20 mM Tris-HCl pH 6.0) and applied to a cation exchange column (HiTrap SP FF, cytiva). The column was washed with 10 column volumes of buffer II (20 mM Tris-HCl pH 6.0, 50mM sodium chloride) and eluted with a linear gradient of 20 column volumes of sodium chloride (from 0.05M to 0.5M). Fractions containing the secreted Gm11437 fragment were pooled, diluted 6-fold with buffer I, and reloaded onto HiTrap SP FF columns equilibrated with buffer II. The column was washed with 10 column volumes of buffer III (20 mM Tris-HCl pH 6.0, 150mM sodium chloride) and eluted with a linear gradient of 20 column volumes of sodium chloride (from 0.15M to 0.3M). Fractions were pooled, concentrated and further purified by size exclusion chromatography in PBS buffer (Superdex 200 Increatase 10/300GL, cytiva). The peak fractions were pooled and concentrated for subsequent analysis.

To purify the incretins expressed and secreted by insect cells, the pFastBac baculovirus system was used to express incretins with 3 XFlag and 6 XHis tags in Hi-5 cells. Conditioned medium was collected 48h after viral infection and applied to equilibration buffer (20 mM NaH) ₂ PO ₄ pH7.4, 500mM NaCl) on a nickel affinity column (HisTrap excel, cytiva). Washing with 20 column volumes of washing buffer (20 mM NaH) ₂ PO ₄ pH7.4, 500mM NaCl,10mM imidazole) and eluted with a 15 column volume linear imidazole gradient from 0.01M to 0.5M. The peak fractions were pooled, 10-fold diluted with a buffer containing 20mM Tris-HCl pH 6.0, and then applied to a HiTrap SP FF column. The column was washed with 5 column volumes of buffer containing 20mM Tris-HCl pH 6.0, 50mM NaCl and a linear gradient of sodium chloride (from 0.05M) over 20 column volumesTo 0.5M) was eluted. Fractions containing the enterosurvivin were pooled, concentrated, and further purified in PBS buffer using a Superdex 200 Incrase 10/300GL column. The peak fractions were pooled and concentrated for subsequent experiments. The incretin-Flag-His purified from Hi-5 cells was used for functional assays in this study.

To purify GST or His-tagged incretins from bacteria, murine incretins were cloned into pGEX-4T-1 vector or pCold-ProS2 for expression in E.coli. Bacteria expressing GST, GST-incretin, his-ProS2 or His-Pro 2-incretin were centrifuged at 7000g and lysed homogenously in a lysis buffer containing 50mM Tris-HCl pH7.4, 150mM NaCl and 1mM PMSF. The lysates were centrifuged at 47850g for 20 min and GST or GST-enterosurvivin supernatants were incubated with glutathione agarose (Pierce Biotechnology) for 2 h at 4 ℃. His-ProS2 and His-Pro 2-enterosurvivin supernatants containing 5mM imidazole were incubated with cobalt affinity resin at 4 ℃ for 2 hours. GST or GST-incretin was eluted with pH7.4 buffer containing 10mM reduced glutathione, 150mM NaCl and 50mM Tris-HCl at 4 ℃. His-ProS2 or His-ProS 2-incretins were eluted with pH7.4 buffer containing 200mM imidazole, 150mM NaCl and 50mM Tris-HCl at 4 ℃. The protein solution was concentrated and further purified using a Superdex 200 Increate 10/300GL column in PBS buffer. The purified protein was stored for subsequent analysis.

To purify OLFR796 expressed in insect cells, OLFR796 and an enterosurvivin binding deficient OLFR796 mutant with a C-terminal 3 xflag and 6 xhis tag (R187D, R195D and E197A) were expressed in Hi-5 insect cells using the pFastBac baculovirus system. Cells were harvested 72 hours after virus infection, lysed with a buffer homogenate containing 20mM Tris-HCl pH 7.5, 50mM NaCl and protease inhibitor and centrifuged at 20216g for 10 minutes. The supernatant was then centrifuged at 68905g for 60 minutes and the membrane fraction was solubilized and precipitated with 2% n-dodecyl- β -D-maltoside and 0.4% sodium cholate at 4 ℃ for 2 hours. After centrifugation at 68905g for 30min, the supernatant was applied to cobalt affinity resin (TALON, takara). The resin contained 20mM Tris pH 7.5, 300mM NaCl, 0.4% n-tendialkyl-beta-D-maltoside, 0.08% sodium cholate, 10mM imidazole and protease inhibitor wash buffer 1 (W1), and again with buffer 2 (W2) containing 20mM Tris pH 7.5, 150mM NaCl, 0.2% n-dodecyl-beta-D-maltoside, 0.04% sodium cholate and protease inhibitor. The eluate was collected and applied to anti-Flag M2 affinity resin (Sigma). The resin was washed with W2 buffer and 200. Mu.g ml of W2 buffer was added ^-1 The FLAG peptide eluted the protein, concentrated and then purified by size exclusion chromatography (Superdex 200 Incrase 5/150 GL, cytiva) in PBS buffer containing 0.2% n-dodecyl- β -D-maltoside and 0.04% sodium cholate. The peak fractions were pooled and concentrated for subsequent analysis.

GST pull-down test

Hi-5 cells transfected with Flag-OLFR796 for 2 days were collected and lysed in cell lysis buffer (50mM HEPES pH7.4, 150mM NaCl,1% Triton X-100) supplemented with protease inhibitor cocktail. The supernatant after centrifugation was mixed with 20. Mu.l glutathione agarose (16101, thermo) and 200ng GST or GST-enterosurvivin recombinant protein purified from bacteria, and rotary-incubated overnight at 4 ℃ for binding reaction. The pellet was washed with cell lysis buffer at high intensity. The glutathione bead bound proteins were eluted, separated on a 12% SDS-PAGE gel and detected by immunoblotting.

Micro calorimetric swimming test

Microcalorimetric phoresis (MST) assay as previously described ²⁷ The process is carried out. The affinity of purified gut survivin-Flag-His to OLFR796 or its mutants was examined using Monolith NT.115 from NanoTemper Technologies. OLFR796 and its mutants were fluorescently labeled according to the manufacturer's protocol, using approximately 100nM of labeled protein per assay. The unlabeled incretin-Flag-His solution was diluted to form an appropriate concentration gradient. The samples were incubated at room temperature for 30 minutes and then loaded into NanoTemper glass capillaries. Measurements were made using 20% of the LED power supply and 40% of the MST power supply. Each affinity assay was repeated 3 times. Kd values for mass action equations were calculated by NanoTemper Analysis software. Changes in thermophoretic motility were expressed as changes in normalized fluorescence (Δ) when analyzedF _norm ) Is defined as F _hot /F _cold 。

Saturation binding assay

The incretin-Flag-His purified from Hi-5 cells was conjugated to biotin using an EZ-link micro Sulfo-NHS-LC-Biotinylation kit (21925, thermo Fisher Scientific). Different doses of biotin-tagged gut pro-survivin-Flag-His were compared with wild-type or Olfr796 with or without over-expression of OLFR796 ^-/- Primary hepatocytes were incubated for 30min at room temperature. Non-specific binding was determined using a 50-fold excess of unbound incretin-Flag-His. Cells were washed three times with PBS, followed by addition of streptavidin-HRP (21130, thermo Fisher Scientific). The absorbance was measured by colorimetry, and the measurement results were normalized by the protein content.

Immunoblotting and immunostaining

In accordance with the previous description ^27、39 The test was conducted. Immunoblotting involves homogenizing cells or mouse tissues in cell lysis buffer. Protein concentration was determined using BCA protein assay kit (Thermo Fisher, 23227). Samples were loaded on SDS-PAGE gels and then transferred to nitrocellulose membranes. Immunoblotting was performed with the corresponding antibody in gelatin buffer (50mM Tris HCl pH7.4, 150mM NaCl,5mM EDTA,0.05% Tween-20). The purchase and dilution of the antibodies were as follows: anti-pATGL (ab 135093,1, 1000), abcam; anti-ATGL (2138s, 1 1000), anti-pAKT (9275s, 1; anti-TUBA 1A (T6199, 1 10000) and anti-FLAG (F1804, 1; anti-G6 PC (a 20193,1, 2000) and anti-PCK 1 (a 2036, 1. Rat anti-incretin (1.

In the cell immunostaining assay, cells were fixed in a 4% Paraformaldehyde (PFA) solution. The non-penetrated cells were blocked with PBS containing 5% BSA, and the penetrated cells were blocked with PBS containing 0.2% Triton X-100 and 5% BSA. The samples were stained with the specific primary antibody overnight in 4 ℃ blocking solution, washed 3 times with PBS, and then incubated with the fluorescent dye for 1 hour in room temperature blocking solution. The samples were incubated in DAPI solution for 10 minutes to stain the DNA, and then coverslipped. Tiled images were obtained using a fluorescence microscope (Zeiss) with the exposure time of each channel for all slides kept constant. Signal intensity was quantified using ImageJ.

To determine the binding of enterosurvivin to the cells, 293T cells and primary hepatocytes were plated on glass coverslips. Cells were incubated with 100nM GST or GST-enterosurvivin for 30min at 37 deg.C, washed 2 times with cold PBS, and immediately fixed with 4% PFA buffer solution. Tissue binding experiments: mouse tissues were dissected, fixed with buffered 4% PFA overnight at 4 deg.C, cryopreserved overnight with 30% sucrose solution, and finally embedded with OCT (Sakura). Frozen tissue sections (8 μm) were incubated with 100nM GST or GST-enterosurvivin for 2 hours at room temperature. For competitive binding, cells and frozen tissue sections were incubated with 100nM GST-enterosurvivin in the presence of 5. Mu.M His-ProS2 or His-ProS 2-enterosurvivin for 2 hours at room temperature. Fixed cells and frozen tissue sections were stained with anti-GST antibody (WH 117206, abclonal) and Alexa Fluor 488 conjugated antibody.

Quantitative PCR

Total RNA was extracted from cells or mouse tissues using a total RNA extraction kit (Omega). The cDNA was obtained using the RevertAID First Strand cDNA Synthesis kit (Thermo). cDNA arrays of cDNA from human tissues were purchased from Origene (HMRT 504). RNA levels were measured with LightCycler 480II (Roche) as described previously ^27，39 . qPCR employed the following primers:

actin-forward primer: 5' GTCCACCCCCGGGAAGGTGA-3

Actin-reverse primer: 5' AGGCCTCAGACCTGGCCATT-3

C17orf 78-forward primer: 5' AACAGCTGCCTGGGATCTTCTTCC-3

C17orf 78-reverse primer: 5' flag TGCTTGACCTTTGGCTCTTTC-3

Cel-Forward primer: 5' CTGGCCCAGACACAAAAGC-3

Cel-reverse primer: 5' GGGAAAACAGGTAATAAGTCTTG-doped 3

Clps-forward primer: 5' ACCAACACCAACTATGGCATCT-3

Clps-reverse primer: 5' CCAGCTAACTGCGTGATCTCA-3

Cpt1 a-Forward primer: 5' CAAAGATCAATCGGACCTAGAC-3

Cpt1 a-reverse primer: 5' CGCCACTCACGATGTTTCTTC-3

G6 pc-forward primer: 5' GTGAATTACCAAAGACTCCCAGGACTG-containing 3

G6 pc-reverse primer: 5' GaTGGAACCAGATGGGAAAGAGGAC-3

Gm 11437-forward primer: 5 'and 3' of TTGGCTCTGGAAGGAGAGTGA-

Gm 11437-reverse primer: 5' TAGGAAGATCAGTAATGACTGGCA-3

Hmgcs 2-forward primer: 5' CCGTATGGGCTTCTGTTCAG-3

Hmgcs 2-reverse primer: 5' AGCTTTGTGCGTTCCATCAG-3

Olfr 796-forward primer: 5' GCCGCAAGGTCTTCTACC-3

Olfr 796-reverse primer: 5' TATGTGATGCTGGCCGTTCC-3

Pck 1-Forward primer: 5' ATGTGGCCAGGATCGAAAGCAAGAC-3

Pck 1-reverse primer: 5' CTTTCATGCATGCCCTGGGAACCTGG-3

Pnlip-forward primer: 5' ACAACAGAAACCCGTATATTAT-3

Pnlip-reverse primer: 5' TGCACACATGTCAGATAGCCAGTT 3

Pnliprp-forward primer: 5' CCCCTCTGTCCTCCTATGAGAGAG-3

Pnliprp-reverse primer: 5' CCATTTTGGGACACCCTTGT

Mass Spectrum (MS)

To determine the cleavage site of Gm11437, the purified Gm 11437N-terminal product was analyzed by electrospray tandem mass spectrometry on a Thermo LTQ Orbitrap instrument, as previously described ^27、39 . Mass spectral proteomics data was stored in a protemexchange database by the PRIDE partner repository (PRIDE partner repositivity), with a data set identifier PXD024577.

Statistical method

Age and weight matched male mice were randomly assigned to the experiments. The number of animals used in all experiments is listed in the corresponding graphic legend. No animals were excluded from the statistical analysis and the investigators were not blinded during the study. All studies were performed in at least three independent experiments. Results are expressed as mean ± s.e.m. Comparisons between different groups were performed using two-tailed unpaired student's t-test or two-factor analysis of variance. P <0.05 was considered statistically significant for the differences. No statistical method is used to predetermine the sample size.

Single transmembrane protein (sTMP) on the cell surface is involved in a variety of cell signaling pathways in response to environmental signals and controlling a variety of biological processes from metabolism, proliferation, apoptosis to immune responses ^6、7 . There are approximately 1300 sstmp on the human cell surface. They act as enzymes, adapters, receptors, co-receptors and ligands to perform their biological functions ^6、7 . Some sTMPs regulate growth and metabolism by shedding and releasing portions of the extracellular domain as a ligand or hormone ^7、8 . For example, epidermal Growth Factor (EGF) is split from pro-EGF and promotes cell proliferation ⁹ . Iridarin (a cytokine (mitokine) secreted from muscle during exercise) is isolated from FNDC5 (fibronectin type III domain containing protein 5) and can promote browning and thermogenesis of white adipose tissue ¹⁰ 。

Incretins are secreted proteins

To determine which sTMP-encoding genes regulate glucose production in hepatocytes, we created a HepG2 cell line stably expressing G6pc-Luc, consisting of a luciferase reporter gene linked to the promoter of G6pc, a gene encoding the rate-limiting enzyme in gluconeogenesis ¹¹ . Cells were transfected with a siRNA library against the 941 human sTMP encoding gene and the control vector RSV-Luc to monitor transfection efficiency and cell viability (fig. 1 a). To monitor the screening process, we chose FOXO1 (forkhead box protein) forkhead boxprotein O1) and CRTC2 (CREB regulated transcriptional co-activator 2) as positive regulators of G6pc-Luc activity, AKT1 and insulin receptor (INSR) as negative regulators ^11-13 (FIG. 1 b). We proceed withTwo such screens gave very good reproducibility (Spearman r = 0.96) and identified four genes that either increased G6pc-Luc activity by more than 1.6 fold or decreased it by at least 40% (fig. 1 b). To test whether the encoded protein affects G6pc-Luc activity by secretion, we performed a transwell assay by overexpressing candidate genes in HEK293T cells and measuring G6pc-Luc activity in mouse primary hepatocytes (fig. 1 a). Overexpression of C17ORF78 (but not the other three genes) increased G6pc-Luc activity (FIG. 1C, FIG. 1 d), indicating that C17ORF78 can affect G6pc-Luc activity through secretion. Similar results were also obtained using the mouse C17orf78 homolog Gm11437 (fig. 1e to 1 g). Furthermore, immunostaining showed that the N-terminus of Gm11437 was extracellular (fig. 1 h).

To determine the amino acid sequence of secreted Gm11437, we over-expressed Gm11437 in insect cells (Sf 9) using a baculovirus expression system and purified the secreted Gm11437 from the cell culture medium (fig. 1i, fig. 1 j). Secreted Gm11437 is highly glycosylated due to its protective effect under starvation conditions, designated as incretin (fig. 1 k). Next, we identified the amino acid sequence of enterosurvivin by mass spectrometry and found that it corresponds to extracellular amino acids 1-191 of Gm11437 (FIG. 1l, FIG. 1 m). Similarly, after overexpression of Gm11437 in HEK293T cells, a highly glycosylated incretin was detected in the culture medium and eliminated by inhibition of the proprotein convertase (fig. 1n, fig. 1 o). Overexpression of furin (but not other proprotein convertases) can significantly increase the levels of incretins (fig. 1 p). In fact, furin may be in Argine ¹⁹¹ The site cleaves Wild Type (WT) Gm11437 but not Gm11437 mutant (K190A/R191A, AA) (FIG. 1 q), which is consistent with the cleavage site of furin 14. Compared with WT Gm11437, the Gm11437/AA mutant lost the stimulatory effect on G6pc-Luc activity in the transwell assay, whereas the stimulatory effect of the enterosurvivin on G6pc-Luc activity was stronger than that of WT Gm11437 (FIG. 1f, FIG. 1G). Similar results were obtained using C17ORF78 (fig. 1C, fig. 1 d). Taken together, these results show that: the incretin is a secreted protein, which is cleaved by furinAnd then released from Gm11437.

Incretin is a fasting inducing hormone secreted mainly by the intestinal tract

To determine the physiological function of incretins, we performed an ELISA analysis and found that plasma incretins levels exhibit diurnal fluctuations with an acute decline as feeding begins (fig. 2 a). In contrast, plasma incretin levels are elevated after fasting. The maximum level (700 pM) was reached after 18 hours of fasting and then decreased after continued fasting (fig. 2 b). Taken together, these results indicate that incretin is a fasting induced hormone.

Gm11437 is highly expressed in the intestine, especially in the proximal intestine (duodenum) (fig. 2 c). It was expressed in the liver in small amounts and was not detected in other tissues. Similar to the tissue distribution of Gm11437, C17orf78 (human homolog of Gm 11437) is highly expressed in the gut (fig. 2 d). To determine which tissues are the major source of circulating enterosurvivin, we constructed mice with tissue-specific knockouts of Gm11437 in either the intestinal tract (IKO) or Liver (LKO) (fig. 2 e). The plasma incretin levels increased from-100 pM to-600 pM in both Wild Type (WT) and LKO mice after overnight fasting, while the fasting-induced increase in plasma incretin levels in IKO mice was abolished (FIG. 2f, FIG. 2 g). Taken together, these results indicate that circulating gut survivin is secreted primarily from the gut during fasting periods.

Incretin for promoting survival of mice

Many mammals adopt adaptive energy conserving survival strategies such as hibernation to reduce core body temperature (Tb) and voluntary activity and increase energy mobilization when challenged by poor food or harsh environmental conditions ^4，5 . Since incretins are induced during fasting, we tested its role in fasting-induced hibernation characterized by a deep hypothermia (Tb 25-35 ℃) lasting several hours, and a decline in exercise and metabolic rate ^4，5 . To study fasting induced hibernation, mice were placed at 21 ℃ and a telemetric temperature probe was implanted. During hypothermia, the frequency of dormancy of IKO mice was reduced, and the spontaneous activity of WT mice was clearSignificantly weaker than IKO mice (fig. 3a to 3 c). Furthermore, IKO mice died more easily during fasting than wild-type mice (fig. 3 d). Surprisingly, IKO mice dosed by intraperitoneal injection of incretin restored hibernating responses and mouse survival (fig. 3 a-3 i). This indicates that: circulating incretins (rather than transmembrane protein Gm 11437) are critical for fasting-induced hibernation and starvation survival in mice. Taken together, these results indicate that the incretins are secreted from the gut and promote survival of mice during fasting.

Gm11437 deficiency impairs fasting-induced metabolism

In response to prolonged fasting, the liver produces glucose by gluconeogenesis and produces ketone bodies as the primary fuel. Mobilization of these energy sources is crucial for adapting to fasting metabolism ⁵ . Therefore, we investigated glucose and lipid metabolism in the liver to investigate how intestinal survivin affected the response of mice to fasting. Gm11437IKO mice showed a decrease in blood glucose and plasma β -hydroxybutyrate levels after overnight fasting (fig. 4a, fig. 4 b), indicating that incretins promote glucose and ketone body production. In addition, the liver acetyl-coa levels were significantly reduced in IKO mice (fig. 4 c), suggesting that incretins promote liver fatty acid oxidation. The reduced expression of gluconeogenic genes (G6 pc and Pck 1), dormancy-associated genes (Pnlip, pnliprp2, clps, cel), ketogenic gene (Hmgcs 2) and gene involved in fatty acid oxidation (Cpt 1 a), and fatty triglyceride lipase (ATGL) activity measured in the liver by phosphorylation of fatty triglyceride lipase (pATGL), further confirmed that IKO caused reduced gluconeogenesis, lipolysis, fatty acid oxidation and ketone body production (fig. 4d, fig. 4 e). The expression of lipolytic genes (Pnlip, pnliprp2, clps) is reduced, which is usually induced by hibernation ^19，21 Further supports the importance of the enterosurvivin on hibernation. The pyruvate tolerance test further confirmed the reduced glucose production capacity (FIG. 4 f). At the same time, as measured by the glucose tolerance test and the insulin resistance test: the insulin sensitivity of IKO mice was enhanced (fig. 4g to fig. 4 i). Taken together, these results indicate that incretins promote hepatic glucose and ketone body production during fasting.

OLFR796 is a receptor for incretin

To determine the target tissue for incretins, we performed binding experiments on frozen tissue sections by incubating purified GST-incretins with different mouse tissues. GST-enterosurvivin provided stronger binding signals in the liver and kidney (fig. 5a, fig. 5 c). This binding was specific, as pre-incubation of His-ProS 2-incretin with sections could completely competitively eliminate the signal in liver and mouse primary hepatocytes (fig. 5a to 5 c). These results were further confirmed by saturation binding studies in mouse primary hepatocytes (fig. 5 d).

Interestingly, pre-treatment with the inhibitor of the G protein-coupled receptor (GPCR), SCH-202676, abolished the effect of incretin on G6pc expression (FIG. 5 f). This indicates that the receptor for incretin is a GPCR. To determine the receptors for incretin, we performed siRNA screening in HEK293T cells. The siRNA library targeted 791 a human GPCR-encoding gene and detected enterosurvivin binding by flow cytometry (fig. 5 f). This led us to identify OR10P1 as a candidate receptor for enterosurvivin, since knockout of OR10P1 almost abolished enterosurvivin binding in HEK293T cells (fig. 5 g). GST pull-down assay showed that GST-enterosurvivin purified from E.coli could interact with OLFR796 (mouse homolog of OR10P 1) in Hi-5 cells (FIG. 5 h). Further microcalorimetric electrophoresis (MST) results showed that incretin binds with high affinity to OLFR796 (Kd =4.29 0.45nm) (fig. 5i to 5 l). To confirm this interaction, we introduced 3 mutations (R187D, R195D and E197A) in the extracellular domain between the

transmembrane domains

4 and 5 of OLFR796, a region previously thought to be likely to have ligand binding activity of olfactory receptors ^22，23 . Compared to wild-type OLFR796, OLFR796 mutant (Mut) showed a significant decrease in affinity for incretin (fig. 5k, fig. 5 l), suggesting that these three amino acids are critical for the binding of OLFR796 to incretin.

To confirm whether OLFR796 is required for incretin function, we constructed OLFR796 using CRISPR-Cas9 technology ^-/- Mouse (FIGS. 6a to 6 a)Fig. 6 d). Dose-dependent binding of enterosurvivin was observed in WT primary hepatocytes, but in Olfr796 ^-/- None of the hepatocytes (fig. 6 e). In Olfr796 ^-/- Addition of WT OLFR796 to hepatocytes restored incretin binding, but the OLFR796 mutant (Mut) that had a defect in the incorporation of the anaplerotic incretin had no effect (fig. 6 e). In summary, all of these in vitro and in vivo binding assays confirmed that OLFR796 is a receptor for incretin.

OLFR796 mediates enterosurvivin-induced metabolic adaptation to fasting

Since OLFR796 is a receptor for incretin, we studied Olfr796 ^-/- Whether the mice are similarly metabolically deregulated to incretin deficient mice. Similar to Gm11437IKO mice, olfr796 ^-/- The mice also showed a decrease in blood glucose, plasma β -hydroxybutyrate and liver acetyl-coa levels after overnight fasting (fig. 7a to 7 c). Further, olfr796 ^-/- Mice also showed reduced gluconeogenesis, decreased expression of genes involved in metabolic adaptation to fasting, and increased insulin sensitivity (fig. 7 d-7 h). Olfr796 gene knock-out resulted in a reduced frequency of occurrence of hibernation, decreased survival and increased autonomic activity (fig. 7j to 7 n). In addition, the incretin potentiating effect in WT mice was in Olfr796 ^-/- Was eliminated in mice (fig. 7j to fig. 7 n). These results gave Olfr796 ^-/- Support of down-regulation of expression of key genes involved in gluconeogenesis, lipolysis, fatty acid oxidation and ketone body production in mice (figure 7 i). Taken together, all these results indicate that Olfr796 ^-/- Mice almost phenotypically mimic incretin deficient mice, while OLFR796 is essential for incretin-induced metabolic adaptation during fasting.

Given the significant effect of the incretin-OLFR 796 signaling axis on fasting induced carbohydrate metabolism, we examined whether this signaling axis plays a role in hyperglycemia in obesity and/or diabetes models. Plasma incretin levels were significantly elevated in obese and diabetic mice in the free-fed state, whereas fasting-induced increases in plasma incretin levels were attenuated in obese and diabetic mice (fig. 8a, fig. 8 b). And is smallThese results are similar in mice, with diabetic patients having higher plasma incretin levels 2 hours after feeding and less incretin production after fasting, compared to normal humans (fig. 8 c). Thus, these results indicate that reducing the effects of gut survivin may improve blood glucose levels in obese and/or diabetic models. In fact, neutralization of enterosurvivin by antibodies decreased the blood glucose levels in WT mice, but Olfr796 was fed to HFD for 16 weeks ^-/- There was no significant effect in the mice (fig. 8 d). Taken together, these results suggest that the incretin-OLFR 796 axis may be a potential target for the treatment of type 2 diabetes.

Discussion of the preferred embodiments

Although it is well known that the intestine, as the major organ for nutrient absorption, regulates the metabolism of different organs during feeding, the role of the intestine in fasting-induced metabolic adaptation remains largely unknown. Here we found that the fasting induced hormone incretin is secreted from the gut and binds to its receptor OLFR796 to promote hibernation and fasting induced energy mobilization enhancement in mice, thereby promoting survival in starvation. Our findings indicate that the gut plays an important role during fasting through incretin-mediated organ-organ communication. Furthermore, our research enriches an ever-expanding list of olfactory receptors that regulate various metabolic processes.

It will be apparent to those skilled in the art that variations and modifications of the present invention can be made without departing from the scope or spirit of the invention. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Reference documents:

1.Small,C.J.&Bloom,S.R.Gut hormones and the control of appetite.Trends Endocrinol Metab 15,259-263,doi:10.1016/j.tem.2004.06.002(2004).

2.Holst,J.J.,Gribble,F.,Horowitz,M.&Rayner,C.K.Roles of the Gut in Glucose Homeostasis.Diabetes Care 39,884-892,doi:10.2337/dc16-0351(2016).

3.Priest,C.&Tontonoz,P.Inter-organ cross-talk in metabolic syndrome.Nat Metab 1,177-1188,doi:10.1038/s42255-019-0145-5(2019).

4.Geiser,F.Metabolic rate and body temperature reduction during hibernation and daily torpor.

Annu Rev Physiol 66,239-274,doi:10.1146/annurev.physiol.66.032102.115105(2004).

5.Jensen,T.L.,Kiersgaard,M.K.,Sorensen,D.B.&Mikkelsen,L.F.Fasting ofmice:a review.Lab Anim 47,225-240,doi:10.1177/0023677213501659(2013).

6.Bugge,K.,Lindorff-Larsen,K.&Kragelund,B.B.Understanding single-pass transmembrane receptor signaling from a structural viewpoint-what are we missingFEBS J 283,4424-4451,doi:10.1111/febs.13793(2016).

7.Pahl,M.C.et al.in eLS(John Wiley&Sons Ltd,Chichester,UK.,2013).

8.Lichtenthaler,S.F.,Lemberg,M.K.&Fluhrer,R.Proteolytic ectodomain shedding of membrane proteins in mammals-hardware,concepts,and recent developments.EMBO J 37,doi:10.15252/embj.201899456(2018).

9.Massague,J.&Pandiella,A.Membrane-anchored growth factors.Annu Rev Biochem 62,515-541,doi:10.1146/annurev.bi.62.070193.002503(1993).

10.Bostrom,P.et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development ofwhite fat and thermogenesis.Nature 481,463-468,doi:10.1038/nature10777(2012).

11.Petersen,M.C.,Vatner,D.F.&Shulman,G.I.Regulation ofhepatic glucose metabolism in health and disease.Nat Rev Endocrinol 13,572-587,doi:10.1038/nrendo.2017.80(2017).

12.Altarejos,J.Y.&Montminy,M.CREB and the CRTC co-activators:sensors for hormonal and metabolic signals.Nat Rev Mol Cell Biol 12,141-151,doi:10.1038/nrm3072(2011).

13.Lin,H.V.&Accili,D.Hormonal regulation of hepatic glucose production in health and disease.Cell Metab 14,9-19,doi:10.1016/j.cmet.2011.06.003(2011).

14.Seidah,N.G.&Prat,A.The biology and therapeutic targeting of the proprotein convertases.Nat Rev Drug Discov 11,367-383,doi:10.1038/nrd3699(2012).

15.Gavrilova,O.et al.Torpor in mice is induced by both leptin-dependent and-independent mechanisms.Proc Natl Acad Sci USA 96,14623-14628,doi:10.1073/pnas.96.25.14623(1999).

16.Gluck,E.F.,Stephens,N.&Swoap,S.J.Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway.Am J Physiol Regul Integr Comp Physiol 291,R1303-1309,doi:10.1152/ajpregu.00232.2006(2006).

17.Kojima,M.et al.Ghrelin is a growth-hormone-releasing acylated peptide from stomach.Nature 402,656-660,doi:10.1038/45230(1999).

18.Swoap,S.J.The pharmacology and molecular mechanisms underlying temperature regulation and torpor.Biochem Pharmacol 76,817-824,doi:10.1016/j.bcp.2008.06.017(2008).

19.Inagaki,T.et al.Endocrine regulation of the fasting response by PPARalphamediated induction of fibroblast growth factor 21.Cell Metab 5,415-425,11

Doi:10.1016/j.cmet.2007.05.003(2007).

20.Owen,B.M.,Mangelsdorf,D.J.&Kliewer,S.A.Tissue-specific actions ofthe metabolic hormones FGF15/19 and FGF21.Trends Endocrinol Metab 26,22-29,doi:10.1016/j.tem.2014.10.002(2015).

21.Zhang,J.,Kaasik,K.,Blackburn,M.R.&Lee,C.C.Constant darkness is a circadian metabolic signal in mammals.Nature 439,340-343,doi:10.1038/nature04368(2006).

22.Buck,L.&Axel,R.A novel multigene family may encode odorant receptors:a molecular basis for odor recognition.Cell 65,175-187(1991).

23.Pilpel,Y.&Lancet,D.The variable and conserved interfaces ofmodeled olfactory receptor proteins.Protein Sci 8,969-977,doi:10.1110/ps.8.5.969(1999).

24.Bellono,N.W.et al.Enterochromaffin Cells Are Gut Chemosensors that Couple to Sensory Neural Pathways.Cell 170,185-198 e116,doi:10.1016/j.cell.2017.05.034(2017).

25.Chang,A.J.,Ortega,F.E.,Riegler,J.,Madison,D.V.&Krasnow,M.A.Oxygen regulation of breathing through an olfactory receptor activated by lactate.Nature 527,240-244,doi:10.1038/nature15721(2015).

26.Griffin,C.A.,Kafadar,K.A.&Pavlath,G.K.MOR23 promotes muscle regeneration and regulates cell adhesion and migration.Dev Cell 17,649-661,doi:10.1016/j.devcel.2009.09.004(2009).

27.Li,E.S.,H.；Chen,L.；Long,A.；Zhang,Y.；Liu,Y.；Jia,L.；Wei,F.；Han,J.；Li T.；Liu,X.；Deng,H.；Wang,Y.OLFR734 mediates glucose metabolism as a receptor of Asprosin.Cell Metab,doi:10.1016/j.cmet.2019.05.022(2019).

28.Shepard,B.D.et al.A Renal Olfactory Receptor Aids in Kidney Glucose Handling.Sci Rep 6,35215,doi:10.1038/srep35215(2016).

29.Mani,B.K.&Zigman,J.M.Ghrelin as a Survival Hormone.Trends Endocrinol Metab 28,843-854,doi:10.1016/j.tem.2017.10.001(2017).

Claims

1. an isolated polypeptide having the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 1.

2. The polypeptide of claim 1, wherein at least one amino acid site of said polypeptide is glycosylated.

3. An isolated nucleic acid encoding the polypeptide of claim 1 or 2.

4. A marker for hyperglycemia which is the polypeptide according to claim 1 or 2.

5. Use of a detection reagent for detecting the amount of the polypeptide of claim 1 or 2 in serum for the preparation of a kit for detecting at least one of: blood sugar, plasma beta-hydroxybutyric acid, ketone body and hepatic acetyl coenzyme A.

6. Use of a first agent in the manufacture of a medicament, wherein the first agent is for promoting incretin and the medicament is for at least one of:

improving hunger tolerance;

reducing insulin sensitivity;

increasing blood glucose and/or plasma beta-hydroxybutyrate content;

promoting the production of glucose and/or ketone bodies and/or hepatic acetyl-coa;

promoting liver fatty acid oxidation;

promoting expression of gluconeogenic gene, hibernating related gene, ketogenic gene and/or fatty acid oxidation related gene;

promoting the activity of fatty triglyceride lipase.

7. The use of claim 6, wherein the first agent comprises at least one selected from:

an incretin or incretin functional fragment;

an agent for overexpression of an incretin or functional fragment of an incretin;

an agent for promoting shedding of incretin from Gm 11437;

an enterosurvivin activator;

an agent for enhancing binding of incretin to OLFR 796;

an agent for over-expressing OLFR 796;

OLFR796 activator.

8. Use according to claim 6, wherein the improvement of tolerance to starvation is achieved by:

promoting gluconeogenesis, lipolysis and/or ketone body production;

promote dormancy caused by fasting;

adaptive energy conserving survival strategies such as hibernation, lowering core body temperature (Tb), and/or reducing autonomic activity and/or increasing energy mobilization.

9. The use of claim 6, wherein the gluconeogenic genes are G6pc and Pck1, the hibernation-related genes are Pnlip, pnlip 2, clps and Cel, the ketogenic gene is Hmgcs2 and/or the fatty acid oxidation-related gene is Cpt1a.

10. The use according to claim 7, wherein the agent that promotes the shedding of incretin from Gm11437 is furin.

11. A pharmaceutical composition, food or health product for improving hunger tolerance comprises a first agent for promoting incretin.

12. A pharmaceutical composition, a food or a nutraceutical product according to claim 11, wherein the first agent is as defined in any one of claims 7or 10.

13. Use of a second agent for inhibiting incretin in the manufacture of a medicament for at least one of:

treating or preventing diabetes;

increase insulin sensitivity;

reducing blood glucose and/or plasma beta-hydroxybutyrate content;

reducing the production of glucose and/or ketone bodies and/or hepatic acetyl-coa;

reducing liver fatty acid oxidation;

reducing the expression of gluconeogenic genes, hibernating-related genes, ketogenic genes, and/or fatty acid oxidation-related genes;

reducing the activity of the fatty triglyceride lipase.

14. The use of claim 13, wherein the second agent comprises at least one selected from:

an agent for neutralizing an incretin or functional fragment of an incretin;

an agent for silencing an incretin or a functional fragment of an incretin;

an agent that inhibits the shedding of incretin from Gm 11437;

an incretin inhibitor;

an agent for inhibiting binding of incretin to OLFR 796;

an agent for silencing OLFR 796;

OLFR796 inhibitors;

OLFR796 mutagen.

15. The use of claim 14, wherein the OLFR796 mutagen causes at least one of the following mutations in OLFR 796: R187D, R195D and E197A.

16. A pharmaceutical composition for treating or preventing diabetes, comprising a second agent for inhibiting incretin.

17. A pharmaceutical composition according to claim 16 wherein the second agent is as defined in claim 14 or 15.

18. A method for increasing tolerance to hunger comprising administering to an individual in need thereof a first agent, wherein the first agent is for promoting incretin.

19. A method of reducing weight comprising administering a first agent to an individual in need thereof, wherein the first agent is for promoting incretin.

20. The method of claim 18 or 19, wherein the individual is administered the first agent after experiencing hunger of at least 18 hours.

21. A method according to claim 18 or 19, wherein the first agent is as defined in claim 7or 10.

22. A method for treating or preventing diabetes, comprising administering to an individual in need thereof a therapeutically effective amount of a second agent, wherein the second agent is for inhibiting incretin.

23. The method of claim 22, wherein the second agent is as defined in claim 14 or 15.

24. The method of any one of claims 18 to 23, wherein the administering is achieved by intraperitoneal injection.

25. A method for screening for an agent that increases tolerance to starvation comprising:

contacting the drug to be tested with a starvation model;

comparing the content of incretins in the starvation model system before and after exposure;

wherein, after contacting, an increase in the level of incretin in the starvation model system indicates that the drug being screened is the target drug.

26. The method of claim 25, wherein the starvation model is a starved small intestinal epithelial cell or mouse model.

27. The method of claim 25, wherein the starvation model is a starved mouse model and the contacting is achieved by administering a test drug to the starvation model by intraperitoneal injection.

28. A method for screening a drug for treating or preventing diabetes, comprising:

contacting the screened drug with an obesity or diabetes model;

comparing the amount of incretin in the model system of obesity or diabetes before and after exposure;

wherein a decrease in the level of said incretin in said obesity or diabetes model system after contact indicates that said drug being screened is a drug of interest.

29. The method of claim 28, wherein the model of obesity or diabetes is a mouse model induced by a high fat diet.