CN110179968B - Application of nucleolin in preparing medicine for improving glycometabolism disorder - Google Patents

Abstract

The invention provides application of nucleolin in preparing medicines for improving glycometabolism disorder, regulating glycogen synthase activity and regulating glycogen synthase phosphorylation level. The invention establishes a high fat diet induced glucose metabolism disorder model and a nucleolin overexpression mouse model, confirms that nucleolin is a molecule related to glucose metabolism, and finds that nucleolin can inhibit phosphorylation of liver glycogen synthase to improve activity of glycogen synthase, further improve glycogen synthesis capacity of liver tissues, enhance glucose tolerance and insulin sensitivity of organisms, participate in regulation and control of glucose homeostasis of the organisms and treatment of glucose metabolism disorder, has the function of resisting glucose metabolism disorder and insulin resistance due to up-regulation of expression, provides a new target for prevention and treatment of type 2 diabetes and early stage diabetes, and can be used for preparation of drug research for early intervention of type 2 diabetes and delay of disease process progress.

Description

Application of nucleolin in preparing medicine for improving glycometabolism disorder

Technical Field

The invention relates to the field of biomedicine, in particular to application of nucleolin in preparing a medicament for improving glycometabolism disorder.

Background

In 2017, the number of diabetics in the whole world exceeds 4.5 hundred million, the rate of the diabetes in China is as high as 10.9 percent, and the number of diabetics in the Chinese country is 1.14 hundred million, which is the first worldwide number. The metabolic syndrome is often characterized by the simultaneous existence of non-alcoholic fatty liver disease, hyperlipidemia, hyperglycemia and the like, and the national health and nutrition examination survey (NHANES 1999-2000) in the United states shows that more than half of diabetics are accompanied by dyslipidemia, wherein about 61.6% of diabetics are accompanied by triglyceride increase and 63.7% of diabetics are accompanied by high-density lipoprotein cholesterol reduction. Meanwhile, about 70% of patients with non-alcoholic fatty liver disease are accompanied by glucose metabolism disorder such as insulin resistance or hyperinsulinemia, and about 44% of them are diagnosed as type 2 diabetes. Early epidemic disease survey finds that the prevalence rate of the 7455 population aged more than or equal to 40 years is up to 35.5 percent and is obviously higher than the prevalence rate of fatty liver alone (4.4 percent), blood sugar rise (8.7 percent) or blood fat disorder (19.0 percent).

The relationship between lipotoxicity and diseases has attracted extensive attention at home and abroad, particularly, researches on the pathogenesis of lipotoxicity and metabolic diseases such as diabetes, metabolic syndrome and the like reveal that the common pathological mechanism of glycolipid metabolic disorders and intervention of lipotoxicity harm become important strategies for preventing and treating metabolic diseases, which also is a great problem bothering clinical for a long time. In recent years, the relationship between lipotoxicity and glycometabolism disorder is considered to be complex, so that common pathological bases are very likely to exist, but at present, the molecular mechanism is unclear, new metabolism-related molecules are to be further discovered, and new targets and new strategies are urgently needed for metabolic diseases such as type 2 diabetes mellitus.

Nucleolin (NCL), also known as C23, is currently known to be the most abundant of several hundred nucleolin proteins, accounting for about 10% of the total nucleolin content. Nucleolin is highly conserved in spine animals, and analogs also exist in animals and yeast. The human nucleolin gene is located in the long arm 12 region of chromosome 2 to the end of the long arm and comprises 14 exons and 13 introns.

Nucleolin has three major domains: the N-terminal is rich in acidic residues, mainly contains glutamic acid and aspartic acid, and can be phosphorylated by various kinases including casein kinase 2 (casein kinase 2, CK2), protein Kinase C (PKC) and cyclin dependent kinase 1 (CDK 1); the central domain comprises four RNA recognition motifs (RNA binding domains, RBDs), and the regulation and control effect of nucleolin on various RNAs is the focus of previous research and relates to the synthesis of pre-ribosome RNA and the stability of messenger RNA; the C-terminal is rich in arginine glycine repeats (Arg-Gly-Gly repeats, RGG), and nucleolin is combined with various proteins in the region, such as urokinase-type plasminogen activator (uPA) and receptor thereof, heparin binding cytokine, lactoferrin and the like; meanwhile, the structural domain plays a key role in ribosome assembly and the nuclear entry process of ribosomal protein.

Nucleolin has a wide intracellular localization, shuttles to the nucleus, cytoplasm and cell membrane, and exerts multiple regulatory effects on DNA, RNA and protein metabolism. In recent years, nucleolin has been found to have the functions of resisting angiogenesis, myocardial ischemia-reperfusion injury and the like, and can assist microorganisms such as HIV and influenza virus to infect hosts, besides the functions of participating in chromosome reconstruction, DNA replication, transcription of RNA Pol I/II, ribosome assembly, cell proliferation and apoptosis and the like. Nucleolin, while found primarily in the nucleus, is a protein with multiple subcellular structural localization and complex functions.

Based on the research base of the prior art, the inventor further discovers a new function of nucleolin on the regulation and control of sugar homeostasis of the body and a role of nucleolin in participating in a sugar metabolism process, provides a new mechanism for the occurrence of metabolic diseases which are mainly characterized by glycolipid metabolic disturbance, and provides a new target and a new strategy for preventing and treating metabolic diseases such as type 2 diabetes mellitus and the like.

However, in the prior art, the main research on nucleolin focuses on the action mechanism of nucleolin for inhibiting the proliferation of tumor cells (such as intestinal cancer, gastric cancer and the like), the action mechanism of nucleolin for inhibiting the apoptosis of myocardial cells of diabetic cardiomyopathy and the like, and the research reports of the action mechanism of nucleolin for improving the sugar metabolism disorder of the body, regulating the activity of glycogen synthase and regulating the phosphorylation level of glycogen synthase are not seen so far.

Disclosure of Invention

In order to solve the problems, the invention provides the action mechanism of nucleolin in improving sugar metabolism disorder of organisms, regulating glycogen synthase activity and regulating glycogen synthase phosphorylation level.

The nucleolin of the invention can be human, murine or other nucleolins. In one embodiment, the nucleolin is murine nucleolin, the amino acid sequence is shown in SEQ ID No.4, and the gene sequence thereof can be a nucleic acid sequence encoding the amino acid sequence, and preferably, the gene sequence is shown in SEQ ID No. 3. In a preferred embodiment, the nucleolin is human nucleolin, the amino acid sequence of which is shown in SEQ ID No.2, and the gene sequence of which can be a nucleic acid sequence encoding the above amino acid sequence, preferably, the gene sequence is shown in SEQ ID No. 1.

Sugar metabolism disorder refers to the phenomenon of blood sugar concentration being too high or too low, including but not limited to abnormal blood sugar concentration due to structural, functional, concentration of hormones or enzymes regulating metabolism of glucose, fructose, galactose, etc., or pathophysiological changes of tissues and organs.

Further, the disorder of sugar metabolism includes disorders of sugar metabolism due to high-fat diet, diabetes, hyperglycemia, hyperlipidemia, non-alcoholic fatty liver, obesity, hypoglycemia, fructose metabolic disorder, glycogen storage disease, galactose metabolic disorder, and pyruvate metabolic disorder.

Preferably, the disorder of glucose metabolism is caused by high fat diet, type 2 diabetes, non-alcoholic fatty liver, obesity.

In one embodiment, nucleolin is used in the pathological progression involved in high fat diet-induced dysfunction of sugar metabolism. Preferably, in the research on the influence of nucleolin on the sugar metabolism function of the body, the mice fed with high-fat diet have obvious sugar metabolism disorder and have the symptoms of early-stage lesion of type 2 diabetes, and the nucleolin expression quantity of the liver of the mice is obviously increased, which shows that nucleolin has certain influence on the sugar metabolism function of the body in the early stage lesion of type 2 diabetes and has certain effect in the pathological process of the sugar metabolism disorder induced by high-fat diet.

Further, the drug can improve glucose tolerance and/or insulin sensitivity of the body, and preferably, can improve glucose tolerance and/or insulin sensitivity in the liver.

In one embodiment, nucleolin is involved in the sugar metabolism process through liver tissue, regulating sugar homeostasis in liver tissue. Preferably, in the research on the influence of nucleolin on the hepatic glucose metabolism function, the liver tissue of the mouse injected with the nucleolin overexpression virus has obviously increased expression level of nucleolin gene, and other metabolism-related tissues, such as muscle, fat and the like, have no obvious change in the expression level of nucleolin gene. Meanwhile, the expression level of nucleolin protein of liver of mice with nucleolin over-expression is also obviously increased.

In one embodiment, nucleolin is used to increase glucose tolerance and insulin sensitivity in the body. In another embodiment, nucleolin is used to increase glucose tolerance and insulin sensitivity in the body induced by a high fat diet, thereby combating high fat diet-induced sugar metabolism disorders.

Preferably, in exploring the effects of nucleolin on body glucose and insulin resistance, it was found that nucleolin overexpressing mice under normal dietary conditions have the strongest glucose and insulin resistance. Meanwhile, under the condition of normal diet, the body sugar steady-state regulation capacity of nucleolin overexpression mice is stronger, and the insulin sensitivity is stronger; under the condition of high fat diet, nucleolin overexpression mice have the same sugar tolerance level and insulin sensitivity as the mice under the normal diet condition. Therefore, nucleolin has the function of improving the glucose and insulin tolerance of the body and further resisting high fat diet-induced sugar metabolism disorder.

In another embodiment, nucleolin is used to regulate body glucose homeostasis by increasing insulin sensitivity in liver tissue, improving insulin resistance and thereby achieving regulation of body glucose homeostasis. Preferably, in the research on the influence of nucleolin on biochemical indexes of body blood, it is found that serum total cholesterol, total triglyceride, low density lipoprotein, high density lipoprotein, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase of nucleolin overexpression mice have no obvious difference, fasting blood glucose and blood glucose after being fed with food are both reduced compared with a control group, and the serum insulin level has no obvious change. Therefore, nucleolin has the capacity of improving the insulin sensitivity in the liver and improving the insulin resistance so as to realize the regulation and control of the sugar homeostasis of the body.

Further, the drug can regulate the sugar homeostasis of the body.

In the field of clinical medicine, under the control of the nervous system, the humoral system and the immune system, the normal body coordinates and moves all organs and systems to maintain the relative steady state of the internal environment together, which is called homeostasis. Glucose is an important energy supply substance of the body, sugar in blood is called blood sugar, and is mostly glucose, most of energy required by activities of cells of various tissues in the body comes from glucose, the maintenance of the blood sugar at a certain level plays an important role in the body, and the regulation of blood sugar balance is also part of the regulation of life activities and is an important condition for maintaining homeostasis.

Further, the medicament may be used for the treatment and/or prevention of diabetes, preferably, the medicament is more suitable for the treatment and/or prevention of type 2 diabetes.

Further, the medicament may increase glycogen synthesizing ability of liver tissue.

In preferred embodiments, the glycogen synthase may be of human or murine origin, or of other origin. In one embodiment, the glycogen synthase is murine glycogen synthase and the amino acid sequence is shown in SEQ ID No. 6. In a preferred embodiment, the nucleolin is human nucleolin, the amino acid sequence of which is shown in SEQ ID No.5, and the gene sequence of which can be a nucleic acid sequence encoding the above amino acid sequence.

In one embodiment, nucleolin regulates body glucose homeostasis by increasing glycogen synthesis capacity in liver tissue. Preferably, in exploring the effect of nucleolin on the ability of the liver to synthesize glycogen, it was found that liver glycogen accumulation was significantly higher in nucleolin overexpressing mice. Therefore, nucleolin can participate and regulate the sugar metabolism function of the body by improving the glycogen synthesis capacity of liver tissues.

In another aspect, the invention also provides the use of nucleolin in modulating glycogen synthase activity, preferably, modulating glycogen synthase activity by increasing glycogen synthase activity.

In another aspect, the invention also provides the use of nucleolin in modulating glycogen synthase phosphorylation levels, preferably inhibiting glycogen synthase phosphorylation levels.

In one embodiment, nucleolin regulates sugar homeostasis by inhibiting glycogen synthase phosphorylation, increasing glycogen synthase activity, and thereby increasing glycogen synthesis capacity of liver tissue. Preferably, in exploring the effects of nucleolin on the liver glycogen synthesis mechanism, it was found that after nucleolin protein overexpression, glycogen synthase phosphorylation levels in liver were significantly reduced (serine 641) without significant differences in glycogen synthase levels. Therefore, nucleolin inhibits glycogen synthase phosphorylation, so that glycogen synthase activity is increased, glycogen phosphorylase activity is reduced, glycogen synthesis capability of mouse liver is further enhanced, and regulation of glucose metabolism by nucleolin is further realized by influencing liver glycogen synthesis.

More preferably, nucleolin can be used for preparing glycogen synthase phosphorylation inhibitor drugs.

Increasing glycogen synthase activity or inhibiting the level of glycogen synthase phosphorylation by increasing the level of nucleolin; increasing the level of nucleolin includes increasing the level of nucleolin protein or increasing the level of nucleolin gene, preferably, overexpressing the nucleolin gene. Reducing glycogen synthase activity by inhibiting the level of nucleolin; inhibiting the level of nucleolin includes knocking out, knocking down the level of nucleolin gene expression.

Further, nucleolin is selected from any one or more of the following: I. nucleolin protein, II, nucleolin gene, III, a vector containing nucleolin gene; preferably, the vector comprising the nucleolin gene is a vector expressing the nucleolin gene, more preferably, an adenoviral vector expressing the nucleolin gene.

Further, the dosage form of the drug is selected from liquid dosage forms; preferably, the liquid dosage form is selected from injections.

The invention can bring the following beneficial effects:

the invention establishes a high fat diet induced sugar metabolism disorder model and a nucleolin overexpression mouse model, confirms that nucleolin is a related molecule of sugar metabolism, confirms that nucleolin can inhibit phosphorylation of liver glycogen synthase to improve glycogen synthase activity, further improves glycogen synthesis capacity of liver tissues, enhances glucose tolerance and insulin sensitivity of organisms, participates in regulation and control of sugar homeostasis of the organisms and improvement of sugar metabolism disorder, and has the effect of resisting sugar metabolism disorder and insulin resistance due to up-regulation of expression. The nucleolin plays a role in participating in glucose metabolism disorder and regulating and controlling glucose homeostasis, supplements the current pathological mechanism of metabolic diseases including type 2 diabetes, can also be used as a new target for preventing and treating type 2 diabetes and pre-diabetes (such as insulin resistance, metabolic syndrome and the like), and further can be used for preparing medicine research for early intervention and disease course progress delay of type 2 diabetes.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a schematic diagram showing the effect of nucleolin on the sugar metabolism function of the body in a model of high fat diet-induced sugar metabolism disorder, wherein (A) the body length and body size of mice, (B) the body weight is compared with bar chart, and (C) nucleolin expression is compared with electrophoresis chart;

FIG. 2 is a schematic diagram showing the effect of nucleolin on hepatic glucose metabolism in a nucleolin overexpression mouse model, wherein (A) the nucleolin overexpression mouse model is constructed, (B) a mouse living body imaging graph, (C) a nucleolin gene expression level histogram, and (D) a nucleolin expression contrast electrophoresis graph;

FIG. 3 is a graphical representation of the effect of nucleolin on body glucose and insulin resistance, wherein (A) IPGTT test profile and (B) IPITT test profile;

FIG. 4 is a bar graph of the effect of nucleolin on biochemical indicators of body blood;

FIG. 5 is a graph showing the effect of nucleolin on the ability of the liver to synthesize glycogen, wherein (A) the mouse liver tissue is subjected to scanning electron microscopy (HE) staining and PAS staining, and (B) the mouse liver glycogen content is shown in a bar graph;

FIG. 6 is a schematic diagram of the effect of nucleolin on the mechanism of liver glycogen synthesis, wherein (A) nucleolin overexpression mouse liver primary cell glycogen metabolism-related molecular protein level electrophoresis diagram and (B) nucleolin interaction protein KEGG analysis diagram are shown.

Detailed Description

In order to more clearly explain the overall concept of the present application, the following detailed description is made of the overall scheme of the present invention by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features have not been described in order to avoid obscuring the invention.

Unless otherwise indicated, in the present specification, the experimental reagents, instruments, materials and the like used in the examples and the sources thereof are as follows:

c57BL/6J mice, supplied by Beijing vitamin River Laboratory Animal Technology Co., ltd., (Ltd.); adenovirus, purchased from Shanghai Ji Kai GeneChemicals, inc.; the Pheno Master high-flux intelligent metabolic analysis system is provided by an endocrine laboratory of Shandong province Hospital; the Mindrary full-automatic biochemical analyzer is provided by an endocrine laboratory of Shandong provincial Hospital; an insulin ELISA assay kit purchased from CUSABIO corporation; the liver/muscle glycogen determination kit is provided by Nanjing institute of bioengineering; hematoxylin-eosin staining (HE) stain available from Solarbio; high Fat Diet (HFD) feed was purchased commercially and formulated as: 1.5% cholesterol, 0.25% sodium cholate, 10% lard, 5% sucrose, 83.25% basal feed; normal diet (CD) feed was purchased commercially and was formulated as: 73.5 percent of corn, 25 percent of wheat bran, 1 percent of cereal flour and 0.5 percent of salt.

Unless otherwise stated, western blotting, in vivo imaging technique for small animals, real-time fluorescent quantitative PCR technique, enzyme-linked immunosorbent assay, HE staining, PAS staining, etc. used in the following examples are carried out by known conventional methods, and thus, they are not described in detail.

Example 1 Effect of nucleolin on the function of carbohydrate metabolism in the body

30 male C57BL/6J mice of 6 weeks old are averagely divided into an experimental group and a control group, wherein the experimental group is fed with 60% high fat diet, the control group is normally fed with normal diet, and a high fat diet induced glucose metabolism disorder model is established for simulating early pathological changes of type 2 diabetes. The body weights of two groups of mice are recorded weekly, the appetite behaviors, feces, hair and the like of the two groups of mice are observed, the mice are sacrificed after 28 weeks, the body lengths, body shapes and body weights of the two groups of mice are compared, and the nucleolin expression quantity in the livers of the two groups of mice is detected by a protein immunoblotting method, wherein the amino acid sequence of nucleolin of the mice is shown as SEQ ID No.4, and the obtained result is shown as figure 1.

As can be seen from fig. 1A and 1B, the mice in the high fat diet group had a significantly longer body length, a more robust body shape, and a higher body weight, and showed early symptoms of type 2 diabetes, as compared to the mice in the normal diet group. As can be seen from FIG. 1C, the liver expression level of nucleolin was significantly higher in the mice of the experimental group on the high-fat diet than that of the control group on the normal diet.

Therefore, the mice fed with high-fat diet have obvious sugar metabolism disorder, have the symptoms of early-stage lesion of type 2 diabetes, and have obviously increased nucleolin expression level of liver, which shows that nucleolin has certain influence on sugar metabolism function of organisms in the early-stage lesion of type 2 diabetes and has certain effect in the pathological process of the sugar metabolism disorder induced by high-fat diet.

Example 2 Effect of nucleolin on hepatic glucose metabolism

30 male C57BL/6J mice of 8 weeks old are taken and averagely divided into an experimental group and a control group, wherein the experimental group is injected with nucleolin which is subjected to adenovirus packaging over-expression by tail vein (subsequently, the experimental group is abbreviated as Ad-NCL experimental group), the control group is injected with unloaded virus (subsequently, the control group is abbreviated as Ncon control group), and the infection unit of each group of virus is 3.0 x 10 ⁸ pfu, a nucleolin overexpression mouse model was constructed, as shown in fig. 2A. Two groups of mice were injected once every 6 days, sacrificed 6 times and fasted without water. Performing living body imaging before the mice are killed by adopting a small animal living body imaging technology, detecting the expression level of nucleolin genes in different tissue organs of two groups of mice by adopting a tissue quantitative PCR technology, and detecting protein expression water of nucleolin by adopting a protein immunoblotting methodIn parallel, nucleolin expression in different tissue organs of two groups of mice was compared, and the results are shown in FIG. 2.

Liver tissue that was successfully infected fluoresced green, as shown in figure 2B, liver tissue from both groups of mice was infected. As can be seen from FIG. 2C, the liver tissue of Ad-NCL mice injected with nucleolin overexpression virus showed significantly increased nucleolin gene expression, while other metabolism-related tissues such as muscle and fat showed no significant change in nucleolin gene expression. As can be seen from FIG. 2D, liver nucleolin protein expression levels were significantly increased in Ad-NCL experimental mice injected with nucleolin over-expressing virus compared to Ncon control injected with unloaded virus.

Therefore, nucleolin is involved in the sugar metabolism process in the body through liver tissues, and then the sugar metabolism function of the body is influenced.

Example 3 Effect of nucleolin on body glucose and insulin resistance

The Ad-NCL experimental group and Ncon control group in example 2 were tested for glucose and insulin resistance in mice of each group by an intraperitoneal glucose tolerance (IPGTT) test and an intraperitoneal insulin tolerance (IPITT) test under normal diet (CD) and High Fat Diet (HFD), respectively, to confirm the regulation of glucose metabolism in the liver of mice by nucleolin.

The treatment method of the IPGTT test comprises the following steps: mice were starved for 18h and then were injected with glucose in the abdominal cavity at a dose of 2g glucose/kg mouse body weight, and the mouse tip blood glucose levels were measured every 30 minutes for 120min to confirm the effect of nucleolin in insulin resistance. The treatment method of the IPITT test comprises the following steps: insulin was injected into the abdominal cavity 4h after the mice were starved, at a dose of 1U insulin/kg mouse body weight, and the insulin level was measured every 5-30min for 90min, to confirm that nucleolin can enhance mouse insulin sensitivity and resist high-fat diet-induced insulin resistance. The results obtained above are shown in FIG. 3.

From the IPGTT test curve in FIG. 3A and the IPITT test curve in FIG. 3B, the glucose and insulin resistance of nucleolin overexpressing mice under normal diet was the strongest. Meanwhile, under the condition of normal diet, compared with the Ncon control group, the body sugar steady state regulation capacity of the mice of the Ad-NCL experimental group is stronger, and the insulin sensitivity is stronger; under the condition of high fat diet, compared with the normal diet condition, the Ncon control group shows impaired glucose tolerance and insulin sensitivity, while the Ad-NCL experimental group shows comparable glucose tolerance level and insulin sensitivity to the normal diet condition.

Therefore, nucleolin has the function of improving the glucose and insulin tolerance of the body under normal diet, and simultaneously improving the glucose tolerance and insulin sensitivity under the induction of high fat diet, thereby resisting the high fat diet-induced glucose metabolism disorder.

EXAMPLE 4 Effect of nucleolin on Biochemical indicators of blood in human body

The mouse Ad-NCL experimental group and the Ncon control group constructed in example 2 were used. Monitoring various indexes related to diet and metabolism of the two groups of mice by adopting a PhenoMaster high-throughput intelligent metabolic analysis system, wherein the indexes comprise water and food consumption, oxygen/carbon dioxide consumption, solid and liquid excreta mass, calorie consumption and the like; the weight was measured once a week, and the feces, hair, etc. of each group of mice were observed, and the weight and wet weight of liver and visceral fat were measured after the mice were treated.

A Mindry full-automatic biochemical analyzer is adopted to respectively detect related indexes of lipid metabolism in serum of two groups of mice, including alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), triglyceride (TG), total Cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and the like.

Serum insulin levels of two groups of mice were detected separately using an insulin ELISA assay kit. The results obtained above are shown in FIG. 4.

The results of biochemical blood index measurements of two groups of mice in fig. 4 show that, compared to Ncon control group, there is no significant difference in serum total cholesterol, total triglycerides, low density lipoproteins, high density lipoproteins, glutamic-pyruvic transaminase, and glutamic-oxalacetic transaminase of Ad-NCL mice in nucleolin overexpression, and that fasting blood glucose and blood glucose after satiety of Ad-NCL mice are all decreased compared to control group, and there is no significant change in serum insulin level.

Therefore, nucleolin has the capacity of improving the insulin sensitivity in the liver and improving the insulin resistance so as to realize the regulation and control of the sugar homeostasis of the body.

Example 5 Effect of nucleolin on the ability of the liver to synthesize glycogen

Liver tissue materials of the Ad-NCL experimental group and the Ncon control group mice constructed in the example 2 are respectively taken, fixed and embedded in paraffin to prepare pathological sections, and the pathological sections are stained by HE (high intensity plasma) to observe the liver steatosis and the inflammation activity under a light microscope, and in addition, the glycogen PAS staining is used for observing the glycogen quantity in liver cells, and the glycogen content of the liver tissue is measured by a liver/muscle glycogen measuring kit. The results are shown in FIG. 5.

Scanning electron microscope images of HE-stained liver pathology sections of two groups of mice in fig. 5A showed no significant difference in liver tissues of Ad-NCL experimental group mice compared to Ncon control group, indicating that nucleolin has no effect on steatosis and inflammatory activity of liver tissues; scanning electron microscope images of pathological liver sections stained with glycogen PAS of two groups of mice in FIG. 5A show that liver glycogen accumulation of the mice in the Ad-NCL experimental group is obviously higher than that of the mice in the Ncon control group, and measurement comparison results of glycogen content of liver tissues of the two groups of mice in FIG. 5B further prove the results.

Therefore, nucleolin can participate and regulate the sugar metabolism function of the body by improving the glycogen synthesis capacity of liver tissues.

Example 6 Effect of nucleolin on the mechanism of hepatic glycogen synthesis

Liver tissue materials of the Ad-NCL experimental group and the Ncon control group mice constructed in example 2 were taken, respectively, and subjected to a primary cell culture experiment of mouse liver. And then preparing a mouse liver tissue homogenate or cell lysis protein, and detecting the influence of the overexpressed nucleolin protein on the expression level of the molecules related to the carbohydrate metabolism of the liver cells by using a protein immunoblotting method. The results are shown in FIG. 6.

As can be seen from FIG. 6, after overexpression of nucleolin, the phosphorylation level of glycogen synthase in liver was significantly decreased (serine 641), and there was no significant difference in glycogen synthase level. Therefore, nucleolin inhibits glycogen synthase phosphorylation, so that glycogen synthase activity is increased, glycogen phosphorylase activity is reduced, glycogen synthesis capability of mouse liver is further enhanced, and regulation of glucose metabolism by nucleolin is further realized by influencing liver glycogen synthesis.

Preferably, glycogen synthase activity is increased or the level of glycogen synthase phosphorylation is inhibited by increasing the level of nucleolin; increasing the level of nucleolin includes increasing the level of nucleolin protein or increasing the level of nucleolin gene, preferably, overexpressing the nucleolin gene. Reducing glycogen synthase activity by inhibiting the level of nucleolin; inhibiting the level of nucleolin includes knocking out, knocking down the level of nucleolin gene expression.

In conclusion, the invention establishes a high-fat diet induced sugar metabolism disorder model and a nucleolin overexpression mouse model, confirms that nucleolin is a molecule related to sugar metabolism, confirms that nucleolin can inhibit phosphorylation of hepatic glycogen synthase and improve glycogen synthase activity, further improves glycogen synthesis capacity of liver tissues, enhances glucose tolerance and insulin sensitivity of organisms, participates in regulation and control of sugar homeostasis of the organisms and treatment of sugar metabolism disorder, and has the effect of resisting sugar metabolism disorder and insulin resistance due to up-regulation of expression. The nucleolin plays a role in participating in glucose metabolism disorder and regulating and controlling glucose homeostasis, supplements the current pathological mechanism of metabolic diseases including type 2 diabetes, can also be used as a new target for preventing and treating type 2 diabetes and pre-diabetes (such as insulin resistance, metabolic syndrome and the like), and further can be used for preparing medicine research for early intervention and disease course progress delay of type 2 diabetes.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art to which the present application pertains. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

<110> Shandong provincial Hospital

<120> use of nucleolin for preparing medicine for improving glycometabolism disorder

<130> JH-SDSL-PI-20180252

<141> 2019-04-29

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2133

<212> DNA

<213> Homo sapiens

<400> 1

atggtgaagc tcgcgaaggc aggtaaaaat caaggtgacc ccaagaaaat ggctcctcct 60

ccaaaggagg tagaagaaga tagtgaagat gaggaaatgt cagaagatga agaagatgat 120

agcagtggag aagaggtcgt catacctcag aagaaaggca agaaggctgc tgcaacctca 180

gcaaagaagg tggtcgtttc cccaacaaaa aaggttgcag ttgccacacc agccaagaaa 240

gcagctgtca ctccaggcaa aaaggcagca gcaacacctg ccaagaagac agttacacca 300

gccaaagcag ttaccacacc tggcaagaag ggagccacac caggcaaagc attggtagca 360

actcctggta agaagggtgc tgccatccca gccaaggggg caaagaatgg caagaatgcc 420

aagaaggaag acagtgatga agaggaggat gatgacagtg aggaggatga ggaggatgac 480

gaggacgagg atgaggatga agatgaaatt gaaccagcag cgatgaaagc agcagctgct 540

gcccctgcct cagaggatga ggacgatgag gatgacgaag atgatgagga tgacgatgac 600

gatgaggaag atgactctga agaagaagct atggagacta caccagccaa aggaaagaaa 660

gctgcaaaag ttgttcctgt gaaagccaag aacgtggctg aggatgaaga tgaagaagag 720

gatgatgagg acgaggatga cgacgacgac gaagatgatg aagatgatga tgatgaagat 780

gatgaggagg aggaagaaga ggaggaggaa gagcctgtca aagaagcacc tggaaaacga 840

aagaaggaaa tggccaaaca gaaagcagct cctgaagcca agaaacagaa agtggaaggc 900

acagaaccga ctacggcttt caatctcttt gttggaaacc taaactttaa caaatctgct 960

cctgaattaa aaactggtat cagcgatgtt tttgctaaaa atgatcttgc tgttgtggat 1020

gtcagaattg gtatgactag gaaatttggt tatgtggatt ttgaatctgc tgaagacctg 1080

gagaaagcgt tggaactcac tggtttgaaa gtctttggca atgaaattaa actagagaaa 1140

ccaaaaggaa aagacagtaa gaaagagcga gatgcgagaa cacttttggc taaaaatctc 1200

ccttacaaag tcactcagga tgaattgaaa gaagtgtttg aagatgctgc ggagatcaga 1260

ttagtcagca aggatgggaa aagtaaaggg attgcttata ttgaatttaa gacagaagct 1320

gatgcagaga aaacctttga agaaaagcag ggaacagaga tcgatgggcg atctatttcc 1380

ctgtactata ctggagagaa aggtcaaaat caagactata gaggtggaaa gaatagcact 1440

tggagtggtg aatcaaaaac tctggtttta agcaacctct cctacagtgc aacagaagaa 1500

actcttcagg aagtatttga gaaagcaact tttatcaaag taccccagaa ccaaaatggc 1560

aaatctaaag ggtatgcatt tatagagttt gcttcattcg aagacgctaa agaagcttta 1620

aattcctgta ataaaaggga aattgagggc agagcaatca ggctggagtt gcaaggaccc 1680

aggggatcac ctaatgccag aagccagcca tccaaaactc tgtttgtcaa aggcctgtct 1740

gaggatacca ctgaagagac attaaaggag tcatttgacg gctccgttcg ggcaaggata 1800

gttactgacc gggaaactgg gtcctccaaa gggtttggtt ttgtagactt caacagtgag 1860

gaggatgcca aagctgccaa ggaggccatg gaagacggtg aaattgatgg aaataaagtt 1920

accttggact gggccaaacc taagggtgaa ggtggcttcg ggggtcgtgg tggaggcaga 1980

ggcggctttg gaggacgagg tggtggtaga ggaggccgag gaggatttgg tggcagaggc 2040

cggggaggct ttggagggcg aggaggcttc cgaggaggca gaggaggagg aggtgaccac 2100

aagccacaag gaaagaagac gaagtttgaa tag 2133

<210> 2

<211> 710

<212> PRT

<213> Homo sapiens

<400> 2

Met Val Lys Leu Ala Lys Ala Gly Lys Asn Gln Gly Asp Pro Lys Lys

1 5 10 15

Met Ala Pro Pro Pro Lys Glu Val Glu Glu Asp Ser Glu Asp Glu Glu

20 25 30

Met Ser Glu Asp Glu Glu Asp Asp Ser Ser Gly Glu Glu Val Val Ile

35 40 45

Pro Gln Lys Lys Gly Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys Val

50 55 60

Val Val Ser Pro Thr Lys Lys Val Ala Val Ala Thr Pro Ala Lys Lys

65 70 75 80

Ala Ala Val Thr Pro Gly Lys Lys Ala Ala Ala Thr Pro Ala Lys Lys

85 90 95

Thr Val Thr Pro Ala Lys Ala Val Thr Thr Pro Gly Lys Lys Gly Ala

100 105 110

Thr Pro Gly Lys Ala Leu Val Ala Thr Pro Gly Lys Lys Gly Ala Ala

115 120 125

Ile Pro Ala Lys Gly Ala Lys Asn Gly Lys Asn Ala Lys Lys Glu Asp

130 135 140

Ser Asp Glu Glu Glu Asp Asp Asp Ser Glu Glu Asp Glu Glu Asp Asp

145 150 155 160

Glu Asp Glu Asp Glu Asp Glu Asp Glu Ile Glu Pro Ala Ala Met Lys

165 170 175

Ala Ala Ala Ala Ala Pro Ala Ser Glu Asp Glu Asp Asp Glu Asp Asp

180 185 190

Glu Asp Asp Glu Asp Asp Asp Asp Asp Glu Glu Asp Asp Ser Glu Glu

195 200 205

Glu Ala Met Glu Thr Thr Pro Ala Lys Gly Lys Lys Ala Ala Lys Val

210 215 220

Val Pro Val Lys Ala Lys Asn Val Ala Glu Asp Glu Asp Glu Glu Glu

225 230 235 240

Asp Asp Glu Asp Glu Asp Asp Asp Asp Asp Glu Asp Asp Glu Asp Asp

245 250 255

Asp Asp Glu Asp Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Pro

260 265 270

Val Lys Glu Ala Pro Gly Lys Arg Lys Lys Glu Met Ala Lys Gln Lys

275 280 285

Ala Ala Pro Glu Ala Lys Lys Gln Lys Val Glu Gly Thr Glu Pro Thr

290 295 300

Thr Ala Phe Asn Leu Phe Val Gly Asn Leu Asn Phe Asn Lys Ser Ala

305 310 315 320

Pro Glu Leu Lys Thr Gly Ile Ser Asp Val Phe Ala Lys Asn Asp Leu

325 330 335

Ala Val Val Asp Val Arg Ile Gly Met Thr Arg Lys Phe Gly Tyr Val

340 345 350

Asp Phe Glu Ser Ala Glu Asp Leu Glu Lys Ala Leu Glu Leu Thr Gly

355 360 365

Leu Lys Val Phe Gly Asn Glu Ile Lys Leu Glu Lys Pro Lys Gly Lys

370 375 380

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

385 390 395 400

Pro Tyr Lys Val Thr Gln Asp Glu Leu Lys Glu Val Phe Glu Asp Ala

405 410 415

Ala Glu Ile Arg Leu Val Ser Lys Asp Gly Lys Ser Lys Gly Ile Ala

420 425 430

Tyr Ile Glu Phe Lys Thr Glu Ala Asp Ala Glu Lys Thr Phe Glu Glu

435 440 445

Lys Gln Gly Thr Glu Ile Asp Gly Arg Ser Ile Ser Leu Tyr Tyr Thr

450 455 460

Gly Glu Lys Gly Gln Asn Gln Asp Tyr Arg Gly Gly Lys Asn Ser Thr

465 470 475 480

Trp Ser Gly Glu Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr Ser

485 490 495

Ala Thr Glu Glu Thr Leu Gln Glu Val Phe Glu Lys Ala Thr Phe Ile

500 505 510

Lys Val Pro Gln Asn Gln Asn Gly Lys Ser Lys Gly Tyr Ala Phe Ile

515 520 525

Glu Phe Ala Ser Phe Glu Asp Ala Lys Glu Ala Leu Asn Ser Cys Asn

530 535 540

Lys Arg Glu Ile Glu Gly Arg Ala Ile Arg Leu Glu Leu Gln Gly Pro

545 550 555 560

Arg Gly Ser Pro Asn Ala Arg Ser Gln Pro Ser Lys Thr Leu Phe Val

565 570 575

Lys Gly Leu Ser Glu Asp Thr Thr Glu Glu Thr Leu Lys Glu Ser Phe

580 585 590

Asp Gly Ser Val Arg Ala Arg Ile Val Thr Asp Arg Glu Thr Gly Ser

595 600 605

Ser Lys Gly Phe Gly Phe Val Asp Phe Asn Ser Glu Glu Asp Ala Lys

610 615 620

Ala Ala Lys Glu Ala Met Glu Asp Gly Glu Ile Asp Gly Asn Lys Val

625 630 635 640

Thr Leu Asp Trp Ala Lys Pro Lys Gly Glu Gly Gly Phe Gly Gly Arg

645 650 655

Gly Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Gly Arg Gly Gly

660 665 670

Arg Gly Gly Phe Gly Gly Arg Gly Arg Gly Gly Phe Gly Gly Arg Gly

675 680 685

Gly Phe Arg Gly Gly Arg Gly Gly Gly Gly Asp His Lys Pro Gln Gly

690 695 700

Lys Lys Thr Lys Phe Glu

705 710

<210> 3

<211> 2124

<212> DNA

<213> Mus musculus

<400> 3

atggtgaagc tcgcaaaggc tggcaaaacc cacggtgagg ccaagaaaat ggctcctcct 60

ccaaaggagg tggaagagga tagtgaagat gaagaaatgt cagaagatga agatgacagc 120

agtggagaag aggaggttgt catccctcag aaaaaaggca aaaaggctac cacaacccca 180

gcaaagaagg tggttgtttc acaaacaaaa aaggctgcag ttcccacacc agctaagaaa 240

gcagctgtga ccccaggcaa aaaggcagta gccacaccag ctaagaaaaa cattacacca 300

gccaaagtca ttccaacacc gggtaagaag ggagctgcac aagcaaaagc gttggtacca 360

actcctggta aaaagggagc tgccactcca gctaaggggg ctaagaacgg taagaatgcc 420

aagaaggaag acagtgatga ggatgaagat gaagaggatg aagatgatag cgatgaggat 480

gaagatgatg aggaagagga tgagtttgag ccaccaatag taaaaggagt gaagccagca 540

aaagcagctc ctgctgctcc tgcctcagag gatgaggaag atgatgagga tgaagatgat 600

gaggaagatg atgatgaaga ggaggaagat gactctgagg aagaagttat ggagatcaca 660

acagccaaag gaaagaaaac tcctgcaaaa gttgttccta tgaaagccaa gagtgtggct 720

gaggaggagg atgatgagga agaggatgaa gatgacgagg atgaggatga tgaggaagag 780

gatgacgaag atgatgatga ggaagaagag gaggaagaac ctgttaaagc agcacctgga 840

aaacggaaga aggagatgac caagcagaaa gaagcccctg aagccaagaa acagaaagta 900

gaaggctcag aaccaactac acctttcaat ctgttcattg gaaaccttaa tccaaacaag 960

tctgttaatg aattaaaatt tgccatcagt gaactttttg ctaaaaatga tcttgctgtt 1020

gtggatgtca gaactggtac aaataggaaa tttggttatg tggactttga gtctgctgaa 1080

gacctagaaa aggccttgga gctcactggt ttaaaagtgt ttggcaatga aattaaacta 1140

gaaaaaccaa aaggaagaga tagtaagaaa gttcgagctg caagaacact tctagccaaa 1200

aacctctctt tcaacatcac tgaggatgaa ttaaaggaag tgtttgaaga cgccatggag 1260

atcagattag tcagccagga tgggaaaagt aaagggattg cttatattga atttaagtct 1320

gaagctgatg cagagaaaaa tttggaagaa aagcaggggg cagaaattga tggacgatct 1380

gtttcactct actatactgg agagaaaggt caaaggcaag agagaactgg aaagaccagc 1440

acttggagtg gtgaatcaaa gactttggtt ttaagtaacc tttcctacag tgcaacaaaa 1500

gaaactcttg aggaagtatt tgagaaagca acttttatca aagtgcccca gaacccacat 1560

ggcaaaccta aagggtatgc atttatagaa tttgcttcat ttgaagatgc taaagaagct 1620

ttaaattcct gtaataaaat ggaaattgag ggcagaacaa tcaggctgga gttgcaagga 1680

tccaattcga gaagtcaacc atccaaaact ctgtttgtca aaggtctgtc tgaggatacc 1740

actgaagaga ccttaaaaga atcatttgag ggctctgttc gtgcaagaat agtcactgat 1800

cgggaaactg gttcttccaa agggtttggt tttgtagact ttaatagtga ggaagatgcc 1860

aaagctgcca aggaggccat ggaagatgga gaaattgacg gaaacaaagt taccttggac 1920

tgggccaaac ctaagggtga aggtggcttt ggtggtcgag gtggaggcag aggaggtttc 1980

ggaggcagag gtggaggcag aggtggaaga ggtggatttg gaggaagagg ccggggaggc 2040

tttggaggta gaggaggctt ccgaggcggc agaggaggag ggggagactt caagccacaa 2100

ggaaagaaga cgaagtttga atag 2124

<210> 4

<211> 707

<212> PRT

<213> Mus musculus

<400> 4

Met Val Lys Leu Ala Lys Ala Gly Lys Thr His Gly Glu Ala Lys Lys

1 5 10 15

Met Ala Pro Pro Pro Lys Glu Val Glu Glu Asp Ser Glu Asp Glu Glu

20 25 30

Met Ser Glu Asp Glu Asp Asp Ser Ser Gly Glu Glu Glu Val Val Ile

35 40 45

Pro Gln Lys Lys Gly Lys Lys Ala Thr Thr Thr Pro Ala Lys Lys Val

50 55 60

Val Val Ser Gln Thr Lys Lys Ala Ala Val Pro Thr Pro Ala Lys Lys

65 70 75 80

Ala Ala Val Thr Pro Gly Lys Lys Ala Val Ala Thr Pro Ala Lys Lys

85 90 95

Asn Ile Thr Pro Ala Lys Val Ile Pro Thr Pro Gly Lys Lys Gly Ala

100 105 110

Ala Gln Ala Lys Ala Leu Val Pro Thr Pro Gly Lys Lys Gly Ala Ala

115 120 125

Thr Pro Ala Lys Gly Ala Lys Asn Gly Lys Asn Ala Lys Lys Glu Asp

130 135 140

Ser Asp Glu Asp Glu Asp Glu Glu Asp Glu Asp Asp Ser Asp Glu Asp

145 150 155 160

Glu Asp Asp Glu Glu Glu Asp Glu Phe Glu Pro Pro Ile Val Lys Gly

165 170 175

Val Lys Pro Ala Lys Ala Ala Pro Ala Ala Pro Ala Ser Glu Asp Glu

180 185 190

Glu Asp Asp Glu Asp Glu Asp Asp Glu Glu Asp Asp Asp Glu Glu Glu

195 200 205

Glu Asp Asp Ser Glu Glu Glu Val Met Glu Ile Thr Thr Ala Lys Gly

210 215 220

Lys Lys Thr Pro Ala Lys Val Val Pro Met Lys Ala Lys Ser Val Ala

225 230 235 240

Glu Glu Glu Asp Asp Glu Glu Glu Asp Glu Asp Asp Glu Asp Glu Asp

245 250 255

Asp Glu Glu Glu Asp Asp Glu Asp Asp Asp Glu Glu Glu Glu Glu Glu

260 265 270

Glu Pro Val Lys Ala Ala Pro Gly Lys Arg Lys Lys Glu Met Thr Lys

275 280 285

Gln Lys Glu Ala Pro Glu Ala Lys Lys Gln Lys Val Glu Gly Ser Glu

290 295 300

Pro Thr Thr Pro Phe Asn Leu Phe Ile Gly Asn Leu Asn Pro Asn Lys

305 310 315 320

Ser Val Asn Glu Leu Lys Phe Ala Ile Ser Glu Leu Phe Ala Lys Asn

325 330 335

Asp Leu Ala Val Val Asp Val Arg Thr Gly Thr Asn Arg Lys Phe Gly

340 345 350

Tyr Val Asp Phe Glu Ser Ala Glu Asp Leu Glu Lys Ala Leu Glu Leu

355 360 365

Thr Gly Leu Lys Val Phe Gly Asn Glu Ile Lys Leu Glu Lys Pro Lys

370 375 380

Gly Arg Asp Ser Lys Lys Val Arg Ala Ala Arg Thr Leu Leu Ala Lys

385 390 395 400

Asn Leu Ser Phe Asn Ile Thr Glu Asp Glu Leu Lys Glu Val Phe Glu

405 410 415

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

420 425 430

Ile Ala Tyr Ile Glu Phe Lys Ser Glu Ala Asp Ala Glu Lys Asn Leu

435 440 445

Glu Glu Lys Gln Gly Ala Glu Ile Asp Gly Arg Ser Val Ser Leu Tyr

450 455 460

Tyr Thr Gly Glu Lys Gly Gln Arg Gln Glu Arg Thr Gly Lys Thr Ser

465 470 475 480

Thr Trp Ser Gly Glu Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr

485 490 495

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

500 505 510

Ile Lys Val Pro Gln Asn Pro His Gly Lys Pro Lys Gly Tyr Ala Phe

515 520 525

Ile Glu Phe Ala Ser Phe Glu Asp Ala Lys Glu Ala Leu Asn Ser Cys

530 535 540

Asn Lys Met Glu Ile Glu Gly Arg Thr Ile Arg Leu Glu Leu Gln Gly

545 550 555 560

Ser Asn Ser Arg Ser Gln Pro Ser Lys Thr Leu Phe Val Lys Gly Leu

565 570 575

Ser Glu Asp Thr Thr Glu Glu Thr Leu Lys Glu Ser Phe Glu Gly Ser

580 585 590

Val Arg Ala Arg Ile Val Thr Asp Arg Glu Thr Gly Ser Ser Lys Gly

595 600 605

Phe Gly Phe Val Asp Phe Asn Ser Glu Glu Asp Ala Lys Ala Ala Lys

610 615 620

Glu Ala Met Glu Asp Gly Glu Ile Asp Gly Asn Lys Val Thr Leu Asp

625 630 635 640

Trp Ala Lys Pro Lys Gly Glu Gly Gly Phe Gly Gly Arg Gly Gly Gly

645 650 655

Arg Gly Gly Phe Gly Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly

660 665 670

Phe Gly Gly Arg Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Arg

675 680 685

Gly Gly Arg Gly Gly Gly Gly Asp Phe Lys Pro Gln Gly Lys Lys Thr

690 695 700

Lys Phe Glu

705

<210> 5

<211> 703

<212> PRT

<213> Homo sapiens

<400> 5

Met Leu Arg Gly Arg Ser Leu Ser Val Thr Ser Leu Gly Gly Leu Pro

1 5 10 15

Gln Trp Glu Val Glu Glu Leu Pro Val Glu Glu Leu Leu Leu Phe Glu

20 25 30

Val Ala Trp Glu Val Thr Asn Lys Val Gly Gly Ile Tyr Thr Val Ile

35 40 45

Gln Thr Lys Ala Lys Thr Thr Ala Asp Glu Trp Gly Glu Asn Tyr Phe

50 55 60

Leu Ile Gly Pro Tyr Phe Glu His Asn Met Lys Thr Gln Val Glu Gln

65 70 75 80

Cys Glu Pro Val Asn Asp Ala Val Arg Arg Ala Val Asp Ala Met Asn

85 90 95

Lys His Gly Cys Gln Val His Phe Gly Arg Trp Leu Ile Glu Gly Ser

100 105 110

Pro Tyr Val Val Leu Phe Asp Ile Gly Tyr Ser Ala Trp Asn Leu Asp

115 120 125

Arg Trp Lys Gly Asp Leu Trp Glu Ala Cys Ser Val Gly Ile Pro Tyr

130 135 140

His Asp Arg Glu Ala Asn Asp Met Leu Ile Phe Gly Ser Leu Thr Ala

145 150 155 160

Trp Phe Leu Lys Glu Val Thr Asp His Ala Asp Gly Lys Tyr Val Val

165 170 175

Ala Gln Phe His Glu Trp Gln Ala Gly Ile Gly Leu Ile Leu Ser Arg

180 185 190

Ala Arg Lys Leu Pro Ile Ala Thr Ile Phe Thr Thr His Ala Thr Leu

195 200 205

Leu Gly Arg Tyr Leu Cys Ala Ala Asn Ile Asp Phe Tyr Asn His Leu

210 215 220

Asp Lys Phe Asn Ile Asp Lys Glu Ala Gly Glu Arg Gln Ile Tyr His

225 230 235 240

Arg Tyr Cys Met Glu Arg Ala Ser Val His Cys Ala His Val Phe Thr

245 250 255

Thr Val Ser Glu Ile Thr Ala Ile Glu Ala Glu His Met Leu Lys Arg

260 265 270

Lys Pro Asp Val Val Thr Pro Asn Gly Leu Asn Val Lys Lys Phe Ser

275 280 285

Ala Val His Glu Phe Gln Asn Leu His Ala Met Tyr Lys Ala Arg Ile

290 295 300

Gln Asp Phe Val Arg Gly His Phe Tyr Gly His Leu Asp Phe Asp Leu

305 310 315 320

Glu Lys Thr Leu Phe Leu Phe Ile Ala Gly Arg Tyr Glu Phe Ser Asn

325 330 335

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

340 345 350

Leu Arg Met His Lys Ser Asp Ile Thr Val Met Val Phe Phe Ile Met

355 360 365

Pro Ala Lys Thr Asn Asn Phe Asn Val Glu Thr Leu Lys Gly Gln Ala

370 375 380

Val Arg Lys Gln Leu Trp Asp Val Ala His Ser Val Lys Glu Lys Phe

385 390 395 400

Gly Lys Lys Leu Tyr Asp Ala Leu Leu Arg Gly Glu Ile Pro Asp Leu

405 410 415

Asn Asp Ile Leu Asp Arg Asp Asp Leu Thr Ile Met Lys Arg Ala Ile

420 425 430

Phe Ser Thr Gln Arg Gln Ser Leu Pro Pro Val Thr Thr His Asn Met

435 440 445

Ile Asp Asp Ser Thr Asp Pro Ile Leu Ser Thr Ile Arg Arg Ile Gly

450 455 460

Leu Phe Asn Asn Arg Thr Asp Arg Val Lys Val Ile Leu His Pro Glu

465 470 475 480

Phe Leu Ser Ser Thr Ser Pro Leu Leu Pro Met Asp Tyr Glu Glu Phe

485 490 495

Val Arg Gly Cys His Leu Gly Val Phe Pro Ser Tyr Tyr Glu Pro Trp

500 505 510

Gly Tyr Thr Pro Ala Glu Cys Thr Val Met Gly Ile Pro Ser Val Thr

515 520 525

Thr Asn Leu Ser Gly Phe Gly Cys Phe Met Gln Glu His Val Ala Asp

530 535 540

Pro Thr Ala Tyr Gly Ile Tyr Ile Val Asp Arg Arg Phe Arg Ser Pro

545 550 555 560

Asp Asp Ser Cys Asn Gln Leu Thr Lys Phe Leu Tyr Gly Phe Cys Lys

565 570 575

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

580 585 590

Ser Asp Leu Leu Asp Trp Arg Tyr Leu Gly Arg Tyr Tyr Gln His Ala

595 600 605

Arg His Leu Thr Leu Ser Arg Ala Phe Pro Asp Lys Phe His Val Glu

610 615 620

Leu Thr Ser Pro Pro Thr Thr Glu Gly Phe Lys Tyr Pro Arg Pro Ser

625 630 635 640

Ser Val Pro Pro Ser Pro Ser Gly Ser Gln Ala Ser Ser Pro Gln Ser

645 650 655

Ser Asp Val Glu Asp Glu Val Glu Asp Glu Arg Tyr Asp Glu Glu Glu

660 665 670

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

675 680 685

His Val Pro His Gly Lys Lys Lys Leu His Gly Glu Tyr Lys Asn

690 695 700

<210> 6

<211> 704

<212> PRT

<213> Mus musculus

<400> 6

Met Leu Arg Gly Arg Ser Leu Ser Val Thr Ser Leu Gly Gly Leu Pro

1 5 10 15

Val Trp Glu Ala Glu Arg Leu Pro Val Glu Asp Leu Leu Leu Phe Glu

20 25 30

Val Ser Trp Glu Val Thr Asn Lys Val Gly Gly Ile Cys Thr Val Ile

35 40 45

Gln Thr Lys Ala Lys Thr Thr Ala Asp Glu Trp Gly Glu Asn Tyr Phe

50 55 60

Leu Ile Gly Pro Tyr Phe Glu His Asn Met Lys Thr Gln Val Glu Gln

65 70 75 80

Cys Glu Pro Thr Asn Asp Ala Val Arg Lys Ala Val Asp Ala Met Asn

85 90 95

Lys His Gly Cys Gln Val His Phe Gly Arg Trp Leu Ile Glu Gly Ser

100 105 110

Pro Tyr Val Val Leu Phe Asp Ile Ser Ser Ser Ala Trp Asn Leu Asp

115 120 125

Arg Trp Lys Gly Asp Phe Trp Glu Ala Cys Gly Val Gly Ile Pro His

130 135 140

His Asp Arg Glu Ala Asn Asp Met Leu Ile Phe Gly Ser Leu Thr Ala

145 150 155 160

Trp Phe Leu Lys Glu Val Thr Asp His Ala Asp Gly Lys His Val Ile

165 170 175

Ala Gln Phe His Glu Trp Gln Ala Gly Thr Gly Leu Ile Leu Ser Arg

180 185 190

Ala Arg Lys Leu Pro Ile Ala Thr Val Phe Thr Thr His Ala Thr Leu

195 200 205

Leu Gly Arg Tyr Leu Cys Ala Ala Asn Ile Asp Phe Tyr Asn Gln Leu

210 215 220

Asp Lys Phe Asp Ile Asp Lys Glu Ala Gly Glu Arg Gln Ile Tyr His

225 230 235 240

Arg Tyr Cys Met Glu Arg Ala Ser Val His Cys Ala His Val Phe Thr

245 250 255

Thr Val Ser Glu Ile Thr Ala Ile Glu Ala Glu His Met Leu Lys Arg

260 265 270

Lys Pro Asp Val Val Thr Pro Asn Gly Leu Asn Val Lys Lys Phe Ser

275 280 285

Ala Val His Glu Phe Gln Asn Leu His Ala Met Tyr Lys Ala Arg Ile

290 295 300

Gln Asp Phe Val Arg Gly His Phe Tyr Gly His Leu Asp Phe Asp Leu

305 310 315 320

Glu Lys Thr Leu Phe Leu Phe Ile Ala Gly Arg Tyr Glu Phe Ser Asn

325 330 335

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

340 345 350

Leu Arg Met His Lys Ser Asn Val Thr Val Val Val Phe Phe Ile Met

355 360 365

Pro Ala Lys Thr Asn Asn Phe Asn Val Glu Thr Leu Lys Gly Gln Ala

370 375 380

Val Arg Lys Gln Leu Trp Asp Thr Val His Cys Leu Lys Glu Lys Phe

385 390 395 400

Gly Lys Lys Leu Tyr Asp Gly Leu Leu Arg Gly Glu Ile Pro Asp Met

405 410 415

Asn Ser Ile Leu Asp Arg Asp Asp Leu Thr Ile Met Lys Arg Ala Ile

420 425 430

Phe Ser Thr Gln Arg Gln Ser Leu Pro Pro Val Thr Thr His Asn Met

435 440 445

Ile Asp Asp Ser Thr Asp Pro Ile Leu Ser Thr Ile Arg Arg Ile Gly

450 455 460

Leu Phe Asn Asn Arg Ala Asp Arg Val Lys Val Ile Leu His Pro Glu

465 470 475 480

Phe Leu Ser Ser Thr Ser Pro Leu Leu Pro Met Asp Tyr Glu Glu Phe

485 490 495

Val Arg Gly Cys His Leu Gly Val Phe Pro Ser Tyr Tyr Glu Pro Trp

500 505 510

Gly Tyr Thr Pro Ala Glu Cys Thr Val Met Gly Ile Pro Ser Val Thr

515 520 525

Thr Asn Leu Ser Gly Phe Gly Cys Phe Val Gln Glu His Val Ala Asp

530 535 540

Pro Thr Ala Tyr Gly Ile Tyr Ile Val Asp Arg Arg Phe Arg Ser Pro

545 550 555 560

Asp Asp Ser Cys Asn Gln Leu Thr Gln Phe Leu Tyr Gly Phe Cys Lys

565 570 575

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

580 585 590

Ser Asp Leu Leu Asp Trp Arg Tyr Leu Gly Arg Tyr Tyr Gln His Ala

595 600 605

Arg His Leu Thr Leu Ser Arg Ala Phe Pro Asp Lys Phe His Leu Glu

610 615 620

Pro Thr Ser Pro Pro Thr Thr Asp Gly Phe Lys Tyr Pro Arg Pro Ser

625 630 635 640

Ser Val Pro Pro Ser Pro Ser Gly Ser Gln Ala Ser Ser Pro Gln Cys

645 650 655

Ser Asp Ala Glu Asp Glu Glu Asp Glu Asp Glu Arg Tyr Asp Glu Glu

660 665 670

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

675 680 685

Asn His Phe Pro Lys Gly Lys Lys Lys Leu His Gly Glu Tyr Lys Asn

690 695 700

Claims (9)

1. Use of nucleolin for the manufacture of a medicament for ameliorating a disorder of glucose metabolism; the glucose metabolism disorder comprises glucose metabolism disorder caused by high fat diet, type 2 diabetes, non-alcoholic fatty liver and obesity, and the drug for improving the glucose metabolism disorder can improve the glucose tolerance and/or insulin sensitivity of a body.

2. Use according to claim 1, wherein the medicament can increase glucose tolerance and/or insulin sensitivity in the liver.

3. The use of claim 1, wherein the medicament modulates sugar homeostasis in the body.

4. The use according to claim 1, wherein the medicament is for increasing glycogen synthesising capacity of liver tissue.

5. The use of claim 1, wherein the medicament modulates glycogen synthase phosphorylation levels.

6. Use according to claims 1-5, wherein nucleolin is selected from any one or more of the following: I. nucleolin protein, II, nucleolin gene, III and a vector containing the nucleolin gene, wherein the vector containing the nucleolin gene is a vector for expressing the nucleolin gene.

7. Use according to claim 6, wherein the vector comprising the nucleolin gene is an adenoviral vector expressing the nucleolin gene.

8. Use according to claims 1-5, wherein the medicament is in a dosage form selected from liquid dosage forms.

9. Use according to claim 8, wherein the liquid dosage form is selected from injections.

Images