WO2008055445A1 - Method and composition for increasing insulin sensitivity - Google Patents

Abstract

The present invention provides a use of SIRT1 protein or agonists or up-regulators thereof for manufacturing a composition which can increase insulin sensitivity. SIRT1 can be used to increase insulin sensitivity by down-regulating expression of protein tyrosine phosphatase 1B, a negative regulator of insulin action.

Description

 Methods and compositions for increasing insulin sensitivity

 Technical field

 The invention belongs to the fields of biotechnology and pharmacy, and in particular relates to a method and composition for increasing insulin sensitivity. Background technique

 The number of people with diabetes worldwide has now increased to 170 million. It is expected that by 2030 this figure will reach 3.6 billion, which is a serious threat to human health. The study found that insulin resistance (IR) is the main pathological feature of type I diabetes, which is caused by the relative lack of insulin secretion and insulin barrier. Therefore, increasing insulin sensitivity by increasing insulin sensitivity, reducing fatty acid oxidative metabolism, and inhibiting glycogen export are an effective way to treat type I diabetes.

 At present, drugs that enhance insulin sensitivity mainly include thiazolidinediones, glucagon receptor antagonists, and biguanide hypoglycemic agents. Thiazolidinediones are a new class of insulin sensitizers discovered in recent years. Representative drugs have troglitazone.

(Troglitazone), Rosiglitazone and Pioglitazone. The target of this class of drugs is the nuclear peroxisome-proliferator-activated receptor Υ (ΡΡΑΙΙγ). However, these drugs often have side effects such as hypoglycemia, weight gain, and impaired liver function while effectively lowering blood sugar and enhancing insulin sensitivity (Yki- rvinen H.

Thiazolidinediones. N Eng J Med 2004 351 : 1106-1118). Glucagon is a polypeptide consisting of 29 amino acids. Its physiological role is to promote the decomposition of hepatic glycogen, xenobiotics and lipolysis. Its receptor antagonists can block the above effects and enhance insulin sensitivity (Oureshi SA et al. A novel glucagons receptor antagonist inhibits

Glucagons-mediated biological effects. Diabetes 2004 53 : 3267-3473). The current study found that glucagon receptor antagonists are mainly vanadium compounds, but these drugs are prone to accumulation in bones, kidneys and liver, and cause adverse reactions such as vomiting and dehydration. The main antipyretic drugs of biguanide are metformin, phenformin and butyl bismuth. The mechanism of action is mainly to strengthen the sensitivity of peripheral tissues to insulin, increase the uptake of glucose by peripheral tissues, and reduce the gluconeogenesis in the liver. Lactic acidosis is a major potential adverse reaction to biguanides, and phenformin and buformin are discontinued due to this serious adverse reaction.

 Therefore, there is still a need in the art to find new drugs that increase insulin sensitivity and have higher biosafety. Summary of the invention

 It is an object of the present invention to provide a novel use of a SIRT1 protein or an agonist or an upregulator thereof.

 In a first aspect of the invention, there is provided the use of a SIRT1 protein or an agonist or an upregulator thereof for the preparation of a composition for increasing insulin sensitivity.

 In another preferred embodiment of the invention, the SIRT1 protein or an agonist or an upregulator thereof is used to prepare a composition for increasing insulin sensitivity in the case of insulin resistance.

 In another preferred embodiment of the present invention, the SIRT1 protein or an agonist or an upregulator thereof is also used to prepare a composition which inhibits transcription or expression of protein tyrosine phosphatase-1B.

In another preferred embodiment of the present invention, the upregulator of the SIRT1 protein is a substance that increases expression of SIRT1 protein. In another preferred embodiment of the invention, the composition is for treating a disease associated with a decrease in insulin sensitivity.

 In another preferred embodiment of the present invention, the diseases associated with decreased insulin sensitivity include, but are not limited to: insulin resistance, type 2 diabetes, hyperinsulinemia, diabetic ketoacidosis, hyperosmolar non-ketone Diabetes coma, lactic acidosis.

 In another preferred embodiment of the present invention, the agonist or up-regulating agent of the SIRT1 protein is selected from the group consisting of: resveratrol or an analogue thereof, such as Butine, Isol iquiritigenin, lacquer Fisetin, tetrahydroxytrans hydrazine (Piceatannol; 3, 4, 3 ', 5 '-tetrahydroxy-trans-styrene), or Chinese medicine jaundice. Preferably, the upregulator of the SIRT1 protein is resveratrol.

 In a second aspect of the invention, a method of screening for a potential substance useful for increasing insulin sensitivity is provided, the method comprising:

 The candidate substance is contacted with a system expressing the SIRT1 protein to detect the effect of the candidate substance on the SIRT1 protein; if the candidate substance can increase the expression of the SIRT1 protein or promote the activity of the SIRT1 protein, it indicates that the candidate substance can be used to improve insulin sensitivity. Potential substance.

 In another preferred embodiment of the present invention, the method further comprises: observing the expression or activity of the protein tyrosine phosphatase-1B in the system, and if the expression or activity of the protein tyrosine phosphatase-1B is decreased, This candidate substance is a potential substance that can be used to increase insulin sensitivity.

 In another preferred embodiment of the invention, the system is selected from the group consisting of: a solution system, a subcellular system, a cell system, a tissue system, an organ system, or an animal system.

 In another preferred embodiment of the present invention, the method includes the following steps:

 (a) In the test group, a candidate substance is added to a system that can express or have expressed the SIRT1 protein, and the expression or activity of the SIRT1 protein is detected, and, in the control group, without adding the candidate substance, The expression or activity of SIRT1 protein can be detected in a system that can express or have expressed SIRT1 protein,

 (b) Comparing the expression or activity of SIRT1 protein in step (a) to the expression or activity of SIRT1 protein in the control group,

 If the expression or activity of the SIRT1 protein in the test group is statistically higher (preferably significantly higher than, for example, 15% higher; more preferably, 30% higher) in the control group, it indicates that the candidate substance is useful for improving insulin sensitivity. Potential substance.

 In a third aspect of the invention, there is provided a substance obtainable by the method described for improving insulin sensitivity. In a fourth aspect of the invention, a composition is provided, the composition comprising:

 (i) an effective amount (eg 0.00001-0.1 g / 60 kg body weight / day) of the SIRT1 protein or its agonist or upregulator;

 (ii) an effective amount (eg 0.0005-0.1 g/60 kg body weight/day) selected from the group consisting of: bismuth diabetes drugs, sulfonylurea diabetes drugs, glucosidase inhibitors, insulin sensitization Drugs, aldose reductase inhibitors, insulinotropic drugs;

 (iii) a pharmaceutically or food acceptable carrier.

In another preferred embodiment of the invention, the biguanide drug includes, but is not limited to, metformin, or phenformin. In another preferred embodiment of the present invention, the sulfonylurea diabetes drug includes, but is not limited to, glibenclamide, glipizide, gliclazide, glibenclamide, glimepiride , or gliclazone.

 In another preferred embodiment of the present invention, the glucosidase inhibitor drug includes, but is not limited to, acarbose, vokibose, or miglitol ( Migkitok).

 In another preferred embodiment of the present invention, the insulin sensitizing drug includes, but is not limited to: ciglitazone

(cigktazone), trogkitazone, rosikititazone, or pioglitazone.

 In another preferred embodiment of the present invention, the aldose reductase inhibitor drug includes, but is not limited to, acestatin, epalrestat, and polasite (ponakrestat) ), or torestat (tokrestat).

 In another preferred embodiment of the present invention, the insulinotropic drug comprises, but is not limited to: repaglinide

(repagkinide), or nategkinide.

 In another preferred embodiment, the composition is in a unit dosage form containing resveratrol 0.00001-0.01 g; more preferably, 0.0001-0.01 g.

 In another aspect, the invention provides a method of increasing insulin sensitivity, the method comprising: upregulating the activity or expression level of a SIRT1 protein in a subject, more preferably in the muscle, liver or fat.

 In another aspect, there is provided a method of increasing insulin sensitivity in a patient, the method comprising: administering to the patient 0.00001-0.01 g/day of SIRT 1 protein or an agonist or an upregulator thereof. In another preferred embodiment, the SIRT1 protein or an agonist or upregulator thereof is resveratrol.

 In another aspect, there is provided a method of inhibiting transcription or expression of a protein tyrosine phosphatase-1B, the method comprising: administering to a patient an effective amount of a SIRT 1 protein or an agonist or an upregulator thereof. More preferably, the patient is administered 0.00001-0.01 g/day of SIRT1 protein or an agonist or upregulator thereof.

 Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

 Figure 1A shows that insulin resistance is produced by C2C12 cells induced by palmitic acid; Figure 1B shows that SIRT1 protein is decreased in insulin-resistant C2C12 cells; Figure 1C is a quantitative representation of B; Figure 1D shows HepG2 cells after glucosamine induction Insulin resistance was produced; Figure 1E shows that SIRT1 protein is decreased in insulin-resistant HepG2 cells; Figure 1F shows a quantitative representation of E-graph; (* p < 0.05; ** p < 0.01, Student's T Test; NS is not significant) .

 Figure 2A shows a photograph of HepG2 cells infected with lentivirus CLentivirus; Figure 2B shows a decrease in lentiviral-mediated SIRT1 expression; Figure 2C shows a decrease in SIRT1 expression leading to a decrease in insulin receptor phosphorylation; Figure 2D shows a decrease in SIRT1 expression Insulin-induced glycogen synthesis; Figure 2E shows that the SIRT1 inhibitor Sirtinol blocks insulin-induced glycogen synthesis. (**, ## p < 0.01, Student's T Test; NS means not significant).

Figure 3A shows that normal up-regulation of SIRT1 protein levels has no effect on glucose uptake; Figure 3B shows that up-regulation of SIRT1 protein levels promotes glucose uptake in insulin resistance; Figure 3C shows up-regulation of SIRT1 protein levels on insulin activation signals The effect (* p < 0.05 compared with no insulin and HSV-SIRT1 virus group; # p < 0.05 compared with the single insulin group; Student's T Test). Figure 4A shows that resveratrol treatment promotes glucose uptake under normal conditions; Figure 4B shows that resveratrol treatment promotes glucose uptake in insulin resistance; Figure 4C shows resveratrol treatment on insulin activation The effect of the signal. (* p < 0.05, compared with no insulin and HSV-SIRT1 virus group; # p < 0.05, # # p < 0.01, compared with the single insulin group. Student's T Test).

 Figure 5 shows that SIRT1 RNAi virus can down-regulate SIRT1 protein levels (* p < 0.05, ** p < 0.01 compared to control virus-free resveratrol-treated group. Student's T Test;).

 Figure 6A shows that up-regulation of SIRT1 protein levels in insulin resistance reduces PTP-1B protein levels; Figure 6B shows that up-regulation of SIRT1 protein levels in insulin resistance reduces PTP-1B mRNA levels; Figure 6C shows reduced resveratrol treatment PTP-1B protein levels; Figure 6D shows that resveratrol treatment reduced PTP-1B mRNA levels; Figure 6E is a quantification of the PTP-1B mRNA detection bands of Figure 6B; Figure 6F shows the PTP for Figure 6D Quantification of the band of -1B mRNA was detected (* p < 0.05; ** p < 0.01 compared to insulin treatment alone. Student ' s T Test).

 Figure 7A shows herpesvirus (HSV)-mediated up-regulation of PTP-1B protein levels; Figure 7B shows that up-regulation of PTP-1B protein reverses the increase in glucose uptake caused by up-regulation of SIRT1 protein levels (* p < 0.05. Compared with the virus-free and insulin-free treatment group; ## p 〈 0. 01, compared with insulin and SIRT1 virus treatment group. Student ' s T Test).

 Figure 8A shows that resveratrol can inhibit hyperlipidemia induced by hyperlipidemia; Figure 8B shows that resveratrol can improve glucose tolerance induced by high fat; Figure 8C shows that resveratrol can improve hyperlipidemia induction Insulin tolerance; Figure 8D shows that resveratrol can reduce high-fat-induced elevation of total cholesterol; Figure 8E shows that resveratrol can reduce high-fat-induced elevation of low-density lipoprotein (*P < 0. 05, ** p < 0. 01, compared with the high fat group).

 Figure 9 shows the effect of different Chinese herbal extracts on the Sirt l reporter gene. detailed description

 After extensive and intensive research, the present inventors demonstrated for the first time that SIRT1 (Sirtuin 1) can increase the sensitivity of insulin, which can be used for the prevention or treatment of abnormal expression of protein tyrosine phosphatase-1B (especially increased expression) or Abnormal activity (especially increased activity), or related diseases caused by decreased insulin sensitivity. The present invention has been completed based on this.

 Specifically, the present inventors have found for the first time that SIRT1 can reduce the expression of protein tyrosine phosphatase-IB (PTP-IB) and can increase insulin sensitivity. More specifically, the inventors have found that SIRT1 does not enhance insulin sensitivity under normal conditions, and insulin sensitivity can be increased only when insulin resistance is produced. This enhances the sensitivity of insulin by up-regulating the level or activity of SIRT1 protein, and does not produce side effects such as hypoglycemia caused by excessive insulin sensitivity, and thus has higher biosafety and lower side effects.

At the same time, the inventors have found that the SIRT1 protein agonist, Resveratrol, can not only activate SIRT1 but also up-regulate the protein level of SIRT1. At the same time, animal experiments have shown that resveratrol can significantly alleviate the symptoms of diabetes, including lowering blood sugar, lowering insulin, cholesterol and low-density lipoprotein levels. Resveratrol is a natural compound found in foods that is safe and can be added directly to food or beverages for oral administration. Moreover, the inventors' dose study found that the effective concentration of resveratrol for diseases related to the prevention and treatment of insulin sensitivity is much lower than that of resveratrol used for the prevention and treatment of tumor and cardiovascular diseases. The lower side of the effective concentration greatly reduces the cost of the product, another Aspects can increase the safety of the product.

 In-depth studies have found that SIRT1 and resveratrol have a role in enhancing the sensitivity of insulin to the main target tissues of insulin, such as fat, muscle and liver. The range of action is wider and it is more beneficial to enhance the sensitivity of insulin in different tissues. And for resveratrol, increasing insulin sensitivity not only does not increase weight, but also has a better weight loss effect.

SIRTl (Sirtuin 1)

SIRT1 is a highly conserved, NAD ⁺ -dependent histone deacetylase that is widely present in organisms. In the past, SIRT1 has attracted extensive attention due to its activity to prolong the lifespan of Drosophila and C. elegans (Blander, G. and Guarente, L. 2004. THE SIR2 FAMILY OF PROTEIN DEACETYLASES. Annual Review of Biochemistry 73: 417-435.).

 In the present invention, the SIRT1 protein used may be naturally occurring, for example, it may be isolated or purified from a mammal. Furthermore, the SIRT1 protein may also be artificially produced, for example, recombinant SIRT1 protein may be produced according to conventional genetic engineering recombination techniques. Preferably, the present invention employs a recombinant SIRT1 protein.

 Any suitable SIRT1 protein can be used in the present invention. The SIRT1 protein includes a full-length SIRT1 protein or a biologically active fragment thereof. Preferably, the amino acid sequence of the SIRT1 protein is substantially identical to the sequence shown by GenBank Accession No.: NM 012238 (PubMed).

 The amino acid sequence of the SIRT1 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The SIRT1 protein or a biologically active fragment thereof comprises a substitution sequence of a portion of a conserved amino acid which does not affect its activity or retains its partial activity. Proper replacement of amino acids is well known in the art and the techniques can be readily implemented and ensure that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al., Molecular Biology of The Gene, Fourth, 1987, The Benjamin/Cummings Pub. Co. P224.

 Any biologically active fragment of a SIRT1 protein can be used in the present invention. Here, the biologically active fragment of the SIRT1 protein means a polypeptide which still retains all or part of the function of the full-length SIRT1 protein. Typically, the biologically active fragment retains at least 50% of the activity of the full length SIRT1 protein. Under more preferred conditions, the active fragment is capable of maintaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% activity of the full-length SIRT1 protein.

The modified or modified SIRT1 protein can also be used in the present invention, for example, a SIRT1 protein modified or modified to promote its half-life, effectiveness, metabolism, and/or protein potency. The modified or modified SIRT1 protein may be a conjugate of a SIRT1 protein, or it may comprise a substituted or artificial amino acid. The modified or modified SIRT1 protein may have little in common with the naturally occurring SIRT1 protein, but may also increase insulin sensitivity in mammals without causing other adverse effects or toxicity. That is, any variation that does not affect the biological activity of the SIRT1 protein can be used in the present invention. Use of SIRT1

 Based on the novel findings of the present inventors, the present invention provides the use of a SIRT1 protein or an agonist or an upregulator thereof for the preparation of a composition for improving insulin sensitivity; or for screening for a substance which increases insulin sensitivity.

 Preferably, the SIRT1 protein or an agonist or upregulator thereof is also used to prepare a composition that inhibits transcription or expression of protein tyrosine phosphatase-18.

 Preferably, the SIRT1 protein, an agonist or an upregulator thereof, or a composition comprising the above ingredients can be used to treat a disease associated with decreased insulin sensitivity. More preferably, the diseases associated with decreased insulin sensitivity include: insulin resistance, type 2 diabetes, hyperinsulinemia, and other related diseases caused by insulin resistance, such as: diabetic ketoacidosis, hyperosmolar nonketosis Diabetic coma, lactic acidosis.

 For example, the SIRT1 protein or an agonist or upregulator thereof can be used to: (i) prepare a drug or food that reduces the expression of protein tyrosine phosphatase-1B; (ii) prepare a drug or food that increases insulin sensitivity (more) Preferably, a medicament or food for increasing insulin sensitivity in the case of insulin resistance is prepared; or (iii) a medicament or food for preventing or treating insulin resistance or insulin resistance-related metabolic diseases is prepared.

 In order to demonstrate the above-mentioned use of SIRT1, the inventors studied C2C12 cells and HepG2 cells as cell models, and found that 1 down-regulating SIR levels can lead to insulin resistance; 2 upregulating SIRT1 protein levels to normal glucose intake and insulin Activation signal has no effect, but can significantly enhance glucose uptake and insulin activation signal in insulin resistance; 3 after resveratrol treatment (ie: increase SIRT1 activity or increase expression), promote cell uptake of glucose, and Increased insulin activation signal; 4 Resveratrol enhances hepatocyte glycogen synthesis requires the involvement of SIRT1. These results demonstrate that SIRT1 protein enhances insulin sensitivity in the context of insulin resistance and can be used to prevent or treat diseases associated with decreased insulin sensitivity.

 In addition, the inventors have also found that up-regulation of SIRT1 protein levels or resveratrol treatment (ie, increased SIRT1 activity or increased expression) can reduce protein tyrosine phosphatase-IB (PTP-IB) in the case of insulin resistance. Protein and mRNA levels. PTP-1B itself is a negative regulator of insulin signaling, and is closely related to the pathogenesis and development of metabolic diseases such as obesity and type 2 diabetes. Studies on PTP-1B knockout mice have shown that PTP- is missing. The insulin sensitivity of 1B mice was significantly increased. Therefore, it was demonstrated that the expression or activity of PTP-1B protein can be down-regulated by using SIRT1 protein, thereby achieving the purpose of improving insulin sensitivity.

 Further, the present inventors induced hyperlipidemia in a C57/BL6 mouse insulin resistance model to study the effects of resveratrol administration (i.e., an increase in SIRT1 activity or an increase in expression) on diabetes-related symptoms such as insulin sensitivity. It was found that resveratrol can inhibit hyperlipidemia induced by hyperlipidemia, improve glucose tolerance induced by high fat, improve insulin resistance induced by high fat, reduce elevated total cholesterol levels induced by high fat, and Reduce high fat-induced elevation of low-density lipoprotein levels. These results demonstrate that the administration of resveratrol, or the increase in SIRT1 expression level or activity, can achieve the goal of alleviating diabetes-related symptoms such as insulin sensitivity.

Agonist or upregulator of SIRT1 and its use Any substance that increases the activity of the SIRT1 protein, maintains the stability of the SIRT1 protein, promotes the expression of the SIRT1 protein, prolongs the effective duration of the SIRT1 protein, or promotes the transcription and translation of SIRT1 can be used in the present invention as an increase in insulin sensitivity. Effective substance.

Preferably, the agonist or up-regulating agent of the SIRT1 protein includes (but is not limited to): resveratrol and resveratrol analogs, such as: butein, isoglycyrrhizin (i _S ol iquiritigenin ), pleatin (f isetin), tetrahydroxytrans hydrazine (Piceatannol; 3, 4, 3 ', 5 '-tetrahydroxystyrene), or traditional Chinese medicine Astragalus (or Astragalus membranaceus extract), the extract is preferably It is an extract of water or an aqueous organic solvent of Astragalus plant).

 Since the agonist or up-regulator of SIRT1 can promote the expression of SIRT1 and/or increase the activity of SIRT1, the agonist or up-regulator of SIRT1 can also regulate the sensitivity of insulin by affecting SIRT1 to achieve prevention or The purpose of treating diseases associated with decreased insulin sensitivity. combination

 The present invention also provides a composition comprising an effective amount (e.g., 0.00001-0.01 g / 60 kg body weight / day; preferably, 0.0001 - 0.01 g / 60 kg body weight / day) of the SIRT1 protein or An agonist (such as resveratrol) or an upregulating agent, and a pharmaceutically or food acceptable carrier.

 The composition of the present invention can be directly used for the prevention or treatment of diseases associated with decreased insulin sensitivity. In addition, it can be used in combination with other therapeutic agents or adjuvants.

 Preferably, the composition contains an effective amount of SIRT1 protein and resveratrol.

 Preferably, the composition further comprises an effective amount (e.g., 0.0005-0.1 g / 60 kg body weight / day; preferably, 0.001 - 0.05 g / 60 kg body weight / day) of a substance selected from the group consisting of: biguanides Diabetes drugs, sulfonylurea diabetes drugs, glucosidase inhibitor drugs, insulin sensitizing drugs, aldose reductase inhibitor drugs, insulinotropic drugs.

Preferably, the biguanide drug includes, but is not limited to, metformin, or phenformin; or the sulfonylurea diabetes drug includes (but is not limited to): glibenclamide, glibenclamide Pyrazine, gliclazide, glibenclamide, glimepiride, or gliclazide; or, the glucosidase inhibitor drug includes (but is not limited to): acarbose , vokibose, or migkitok; or, the insulin sensitizing drugs include, but are not limited to: ciglitazone (cigkt _azone ), troglitazone ( t _r0 gkit _azone), rosiglitazone (rosigkitazone), or pioglitazone (piogkitazone) or the aldose reductase inhibitor drugs include (but are not limited to): alrestatin (akrestain), epalrestat He (inkrestat), borakrestat, or tokrestat; or, the insulinotropic drug includes (but is not limited to): repagkinide, or Nategkinide.

 Generally, these materials can be formulated in a non-toxic, inert, andpharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.

As used herein, the term "contains" means that the various ingredients can be used together in the mixture or composition of the invention. Therefore, the terms "consisting essentially of . . . and consisting of " . . . are included in the term "contains". As used herein, the term "has By "effective" or "effective dose" is meant to be functional or active against humans and/or animals and acceptable to humans and/or animals, as used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans. And/or mammals without excessive adverse side effects (eg, toxicity, irritability, and allergies), ie, substances having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to administration of a therapeutic agent. Carriers, including various excipients and diluents. The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). The pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

 The compositions of the present invention comprise a safe and effective amount of a SIRT1 protein and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched with the administration mode, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

 The composition of the present invention can also be administered as a food or dietary supplement directly or added to other foods. Preferably, the "food-acceptable carrier" is selected from the group consisting of: a filler, a disintegrant, a lubricant, a glidant, an effervescent | J, a flavoring agent, a coating material, a dietary product, a fu Forming agent, or slow/controlled release agent.

 As a preferred mode of the present invention, the composition is a unit dosage form, and the "unit dosage form" refers to a dosage form required for preparing a composition of the present invention in a single administration for convenience of administration, including but not limited to various solid dosage forms. (such as tablets), liquid agents, capsules, sustained release agents. 0001-0. 01克。 The unit dosage form composition containing resveratrol 0. 00001-0. 01 gram; more preferably 0. 0001-0. 01 gram. Methods for increasing insulin sensitivity and methods of administration

 The present invention provides a method of increasing insulin sensitivity (e.g., preventing or treating a disease associated with decreased insulin sensitivity), comprising administering to a subject an effective amount of a SIRT1 protein or a system (e.g., a cell or a virus) capable of expressing a SIRT1 protein. When used to treat a disease associated with decreased insulin sensitivity in humans, recombinant SIRT1 protein is preferably employed.

 It will be appreciated that when used to treat a disease associated with decreased insulin sensitivity in a mammal, the effective dose of SIRT1 used may vary with the severity of the subject to be treated. The specific circumstances are determined according to the individual condition of the subject, which is within the range judged by the skilled physician or dietitian.

After the use of the SIRT1 protein is known, the SIRT1 protein or its encoding gene, or a pharmaceutical composition thereof, can be administered to a mammal using a variety of methods well known in the art. Preferably, it can be carried out by means of gene therapy, for example, the SIRT1 protein can be directly administered to the subject by means such as injection; or the expression unit carrying the SIRT1 gene (such as an expression vector or a virus, etc.) can be carried out by a certain route. Delivery to the target and expression of the active SIRT1 protein. As an embodiment of the present invention, the SIRT1 protein may be directly administered to a mammal (such as a human), or the gene encoding the SIRT1 protein may be cloned into a suitable vector (such as a conventional pronucleus or a conventional method). In a eukaryotic expression vector, or a viral vector such as a herpesvirus vector or an adenoviral vector, the vector is introduced into a cell which expresses the SIRT1 protein, and the cell expresses the SIRT1 protein. Expression of the SIRT1 protein in vivo can be achieved by introducing an appropriate amount of the cells into the appropriate part of the mammalian body.

 Preferably, the gene encoding SIRT1 or the vector carrying the gene can be introduced into a target cell or a target tissue by a conventional method to effect expression of the SIRT1 protein. The target cells include, but are not limited to, muscle cells, liver cells, fat cells, and the like; the cells can be transferred into appropriate parts of the mammalian body.

 Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the SIRT1 protein encoding gene and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

 Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Vectors comprising the appropriate gene sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.

 The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf 9; CH0, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.

 When the gene is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of a gene.

 It will be apparent to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

 The obtained transformant can be cultured by a conventional method to express the protein of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction), and the cells are cultured for a further period of time.

The recombinant protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. in case The recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic bacteria, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 The present inventors have also found in the research that some SIRT1 up-regulating agents such as resveratrol are not ideal for higher insulin sensitivity when used for improving insulin sensitivity, and the effect at low doses is ideal. Therefore, as a preferred mode of the present invention, an agonist or up-regulating agent of SIRT1 is administered to a patient, and the up-regulating agent of SIRT1 is resveratrol, preferably, the dose is 0.00001-0.01 g/60 kg. Body weight/day; more preferably, 0.0001 to 0.01 g / 60 kg body weight / day.

 From the experimental results, the concentration of resveratrol administered at about 2.5 mg/kg/d or this concentration is better for rats. According to the general conversion law, the conversion to human consumption is better at about 0.0001-0.01g/60kg/d. Methods for screening potential substances that increase insulin sensitivity

 After the use of the SIRT1 protein is known, various methods well known in the art can be used to screen for substances that increase insulin sensitivity.

 In a preferred form of the invention, a method of screening for a substance that increases insulin sensitivity is provided, the method comprising:

 The candidate substance is contacted with a system expressing the SIRT1 protein to detect the effect of the candidate substance on the SIRT1 protein; if the candidate substance can increase the expression of the SIRT1 protein or promote the activity of the SIRT1 protein, it indicates that it is a potential for improving insulin sensitivity. substance.

 More preferably, the method further comprises: observing the expression or activity of protein tyrosine phosphatase-1B in the system, and if the expression or activity of protein tyrosine phosphatase-1B is decreased, indicating that the candidate substance is usable Potential substances that increase insulin sensitivity.

 In the present invention, the system includes, but is not limited to, a solution system, a subcellular system, a cell system, a tissue system, an organ system, or an animal system. The system may contain SIRT1 for adding candidate substances therein to observe the influence of the candidate substance on SIRT1; or, the system may contain both SIRT1 and PTP-1B for adding candidate substances therein, The effects of candidate substances on SIRT1 and PTP-1B were observed.

 These initially screened materials can form a screening library so that one can ultimately screen out substances that are useful for increasing insulin sensitivity.

 Accordingly, the present invention also encompasses a substance obtained by the screening method described, which substance can be used to increase the sensitivity of insulin. The main advantages of the invention are:

(1) The present invention demonstrates for the first time that SIRT1 can be used to increase insulin sensitivity, revealing that SIRT1 can down-regulate the negative regulator of insulin signaling, PTP-1B, and provides an effective new target for the prevention or treatment of diseases associated with decreased insulin sensitivity. . (2) The present inventors also confirmed that SIRT1 does not enhance insulin sensitivity under normal conditions, and can increase insulin sensitivity only when insulin resistance is produced, and thus has higher biosafety and lower side effects. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, which do not specify the specific conditions, are usually as described in the General Conditions, such as the Guide to Molecular Cloning (J. Sambrook, DW Russell, New York: Cold Spring Harbor Laboratory Press). Conditions, or in accordance with the conditions recommended by the manufacturer.

 I. General materials and methods

 1, cell culture

C2C12 cells (purchased from ATCC, USA) were cultured in DMEM medium (high sugar containing 10% fetal bovine serum) at 37 ° C, 5% C0 ₂ . When the cells were grown to 50-60%, the plates were plated. After the cells were over, they were induced to differentiate for 4 days in DMEM medium containing 2% horse serum, i.e., differentiated into mature muscle cells, and used in the following experiments.

HepG2 cells (purchased from ATCC, USA) were cultured in DMEM medium (high sugar containing 10% fetal bovine serum) under the conditions of 37 ° C, 5% C0 ₂ . When the temperature is 70-80%, the seed plate is digested with trypsin and used.

 2, insulin resistance induction

 C2C12 cells were induced with high glucose DMEM containing palmitic acid (7.5 mM) for 18 h to induce insulin resistance. HepG2 cells were induced with glucosamine (18 mM) low-glucose serum-free medium for 16 h. Produces insulin resistance.

 3. Glucose transport assay

The differentiated C2C12 cells were cultured in 24-well plates (purchased from Corning, USA) containing low-glucose serum-free DMEM culture medium, starved for 3 h, 100 nM insulin was stimulated for 20 min, and 0.5 ³ H labeled deoxyglucose was added per well. Incubate for 5 min, aspirate the culture, place the cell plate on ice, and wash 3 times with PBS.

 Add 200 μM ΐ 0.2 M NaOH per well, collect the cell lysate, and measure the radioactivity using a liquid scintillation meter.

 4, glycogen synthesis determination

HepG2 cells were cultured in 24-well plates, starved for 16 h in low-sugar serum-free DMEM medium, cultured with 100 nM insulin and/or 0.5 μα ³ Η labeled glucose for 3 h, and the culture solution was aspirated, washed twice with PBS, and 200 per well. μ 1 30% NaOH, collect the cell lysate and incubate at 80 ° C for 30 min, centrifuge, add 2 volumes of absolute ethanol, mix, and let stand at -20 ° C overnight.

 After standing, the cells were frozen and centrifuged, the supernatant was discarded, washed with absolute ethanol, centrifuged, and repeated twice. The final precipitate was air-dried, 0.1 N HCl was dissolved, and the radioactivity was measured by a liquid scintillation meter.

 5, protein Western blot

 Add appropriate amount of lysate to the cell culture plate, lyse at 100 ° C for 5 min, and centrifuge for use. Take appropriate amount of protein sample for

Proteins were separated by SDS-PAGE. The protein was electrotransferred to a nitrocellulose membrane and blocked with TBS containing 5% skim milk powder for 1 h, and various corresponding antibodies (anti-Sirtl antibody (purchased from Upstate, USA) and anti-Tubulin antibody (purchased from the United States) were added. Sigma)), overnight at 4 °C, after washing the membrane, add horseradish peroxidase-labeled secondary antibody, shake gently for 1 h at room temperature, wash thoroughly, and react with chemiluminescence reaction kit (purchased from PIERCE, USA) for 2 min. Immediately exposed to X-ray film, and then quantitatively analyzed by laser scanner after washing. 6. Reverse transcription polymerase chain reaction (RT-PCR)

 6.1 Primer Design and Synthesis: Primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.

 Actin (; fragment size is 444 bp):

 Positive strand: 5'ATCACTGCCACCCAGAAGAC'3 (SEQ ID NO: 1),

 Anti-chain: 5'ATGAGGCCACCACCCTG'3 (SEQ ID NO: 2);

PTP-1B (fragment size is 384bp) _:

 Positive strand: 5'CTGACACCTGCCTCTTACTG'3 (SEQ ID NO: 3),

 Reverse chain: 5'CACTTGACTGGGCTCTGC'3 (SEQ ID NO: 4).

 6.2 Total RNA extraction and reverse transcription: Total RNA was extracted and reverse transcribed using a conventional Trizol Reagent.

6.3 PCR amplification: reaction system is 50 μl, 10 mmol/L ldNTP 1 μΐ; 25 mmol/L MgCl ₂ 4 μ1, 10× buffer 5 μ1, 3 'primer 0.5 μ1, 5' primer 0.5 μ1, cDNA 4 μ1, Taq Enzyme 2 U, deionized water to 50 μΐ, reaction conditions: pre-denaturation at 95 °C for 5 min, into the cycle, (95 °C denaturation for 45 s, according to PTP1B: 65 °C; Actin: 57 °C temperature annealing 45 s, extending at 72 °C for 45 s), where PTP1B is 30 cycles and GAPDH is 25 cycles. The amplified product was electrophoresed on a 1.5% agarose gel, and the specific band of the amplified product was observed under a UV transilluminator. The product was subjected to image analysis by a gel imaging and analysis system, with Actin as an internal reference, and the relative level of expression was expressed by the absorbance of the PTP1B band and the Actin ratio.

 7, SIRT1 and PTP-1B overexpression

 7.1 PTP-1B gene cloning

 Design primers:

 Positive strand (SEQ ID NO: 5):

 5 ' GGAT ACGCGT ATGGAGATGGAGAAGGAGTTCGAG 3 ';

 Anti-chain (SEQ ID NO: 6):

 5' GGAAGTCGACAGTGCTCCCAGTCTGTCAGTGAA 3 '

 Total RNA extraction and reverse transcription: The appropriate amount of C2C12 cells was extracted and reversed with Trizol Reagent.

PCR amplification: reaction system is 50 μl, 10 mmol/L ldNTP 1 μΐ; 25 mmol/L MgCl ₂ 4 μ1, 10× buffer 5 μ1, 3 'primer 0.5 μ1, 5' primer 0.5 μ1, cDNA 4 μ1, Taq enzyme 2 U, deionized water to 50 μΐ, reaction conditions: pre-denaturation at 95 °C for 5 min, into the cycle, (denaturation at 95 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s;), 30 Cycles. The amplified product was recovered by gelatinization, digested with Mlul and Sail, and ligated with the same enzyme-cut pHSVPrPUC vector (purchased from Clontech, USA), and the product was coated with a plate, and cloned and identified.

 7.2 Packaging of PTP-1B herpesvirus (HSV)

 The virus was packaged with 2-2 cells (purchased from Harvard Medical School) and the process was as follows:

The PTP1B-containing vector obtained in the appropriate step 7.1 was introduced into 2-2 cells by lipofection method (Lipofactamine 2000), and after 24 hours of culture, the helper virus helper (purchased from Harvard Medical School) required for virus packaging was added, and the culture was continued. Cell change Round, the virus was collected by hypotonic method, and the virus collected for the first time was added to the 2-2 cell expansion culture, and the virus (HSV-PTP-1B) was collected in the same manner, frozen at -80 ° C, and used.

 7.3 SIRT1 gene cloning

 The SIRT1 cDNA fragment was digested with BamHI from the plasmid pBabepuro-SIRTl (purchased from Harvard Medical School) and inserted into the BamHI digestion site of pCMV-Tag 3A (purchased from Stratagene, USA).

 7.4 Packaging of SIRT1 herpesvirus

 The packaging process of SIRT1 herpesvirus is the same as that of PTP-1B herpesvirus. The medium containing SIRT1 obtained in step 7.3 is introduced into 2-2 cells by lipofection method (Lipofactamine 2000;), and cultured for 24 hours. The required helper helper, continue to culture, wait for the cells to round, use the hypotonic method to collect the virus, add the first collected virus to the 2-2 cell expansion culture, and collect the virus (HSV-SIRT) in the same way, -80 °C Freeze, spare.

 7.5 SIRT1 and PTP-1B overexpression detection

 The above HSV virus was added to C2C12 cells, and the expression efficiency was detected by Western blot after 36 h infection.

 8. SIRT1 RNA Intervention (RNAi)

 References (Picard F et al. Sirtl promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature 2004 17: 771-776) The following two oligonucleotides were synthesized to prepare SIRT1 RNAi:

 5'-GATC

TTTTGGAAA-3' (SEQ ID NO: 7);

 5 ' - AGCTTTTCC AAAAAAGA

ACTTCATCG-3' (SEQ ID NO: 8).

 The above two oligonucleotides were mixed and annealed and ligated with the lentiviral expression vector pLentiLox 3.7-H1 (purchased from Harvard Medical School;) (via BamHI and Xhol cleavage sites;), the obtained plasmid and HIV-1 packaging Plasmid Δ8.9 and VSVG plasmids (both purchased from Harvard Medical School) were co-transformed into 293T cells (purchased from ATCC, USA). After 48 h, the supernatant containing lentivirus was collected and frozen at -80 °C. , spare.

 The sequence of the RNAi (Luc RNAi) oligonucleotide used to prepare the control virus luciferase is as follows:

 5'-GATCCGTAGCGCGGTGT

TTTTGGAAG-3' (SEQ ID NO: 9);

 5 ' -TCGACTTCC AAAAAAGT

CGCGCTACG-3 ' (SEQ ID NO: 10).

 The packaging process is the same as the SIRT1 RNAi process.

 The SIRT1 RNAi intervention experiment was performed in HepG2 cells, and the cells were infected with the lentivirus prepared above, and detected by Western.

 9, SIRT1 inhibitor treatment

The SIRT1 inhibitor Sirtinol (50 μΜ) was treated with HepG2 cells for 12 h, and glycogen synthesis was determined by the aforementioned method. 10. Animal experiment C57/BL6 mice were modeled with a high-fat-induced insulin resistance model, and resveratrol was fed while fed a high-fat diet.

(Resveratrol) administration (concentration: 2.5 mg/kg/day;).

 Glucose tolerance (GTT), insulin resistance (ITT), serum total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL) levels were measured four weeks later.

II. Example

 Example 1 SIRT1 protein levels are reduced in the case of insulin resistance

 1. Using C2C12 as a cell model

 According to the aforementioned method, C2C12 cells which are insulin-induced after palmitic acid induction were prepared, and the expression of SIRT1 protein in the cells was measured. The results are shown in Figures 1A-1C.

 Figure 1A shows the glucose transport (relative glucose uptake) of C2C12 cells by the aforementioned glucose transport assay. The results show that C2C12 cells are insulin-induced after palmitate induction (SP: in the absence and presence of insulin) , the relative glucose intake changes are not significant).

 Fig. 1B shows the results of measuring the expression of SIRT1 protein by the aforementioned Western Blot method, in which SIRT1 protein was decreased in insulin-resistant C2C12 cells; Fig. 1C is a quantitative representation of Fig. 1B. In the figure, tubulin (tubulin) is an electrophoresis load control.

 Thus, it can be seen that the expression level of SIRT1 protein is decreased in insulin-resistant C2C12 cells.

 2. Using HepG2 as a cell model

 HepG2 cells producing insulin resistance induced by glucosamine were prepared according to the aforementioned methods, and the expression of SIRT1 protein in the cells was measured. The results are shown in Figures 1D-1F.

 Figure ID shows the glycogen synthesis of C2C12 cells by the aforementioned glycogen synthesis assay method, showing that the cells are insulin-induced after glucosamine induction (i.e., glycogen synthesis is not significantly changed in the absence and presence of insulin) .

 Figure 1E shows the results of SIRT1 protein expression assay using the Western Blot method described above, showing that SIRT1 protein is decreased in insulin resistant HepG2 cells; Figure 1F is a quantified representation of Figure 1E.

 Thus, it can be seen that the expression level of SIRT1 protein is decreased in insulin-resistant HepG2 cells. Example 2 Downregulation SIRT1 can cause insulin resistance

 According to the aforementioned method, HepG2 cells were infected with the lentivirus prepared as described above, and RNA interference was intracellularly SIRT1, resulting in a decrease in SIRT1 protein expression.

 Lentivirus infection of HepG2 cells for 72 h, both control virus and SIRT1 RNA interfering virus can express fluorescent protein, so the expression of fluorescent protein was observed to judge the efficiency of lentiviral infection. The fluorescence of HepG2 cells after lentivirus infection is shown in Figure 2A. The results indicate that lentivirus infection of HepG2 cells is highly efficient, and the control virus is Luc RNA interference.

 Figure 2B shows the results of Western Blot detection of intracellular SIRT1 protein after RNA interference. As can be seen from Figure 2B, lentiviral-mediated RNA interference results in a significant decrease in SIRT1 protein expression.

Figure 2C shows phosphorylation of insulin receptors after RNA interference (antibody and phosphorylation of insulin receptors used in this process) The antibodies to the insulin receptor were purchased from Western Blot of Cell Signaling, USA. Western detection showed that decreased expression of SIRT1 resulted in decreased levels of insulin receptor phosphorylation.

 As can be seen from Fig. 2D, the change in glycogen synthesis in the SIRT1 expression-reduced group in the presence and absence of insulin was not significant. Thus, a decrease in SIRT1 expression prevents insulin-induced glycogen synthesis (in Figure 2D, white bars indicate untreated cells, gray bars indicate Luc-RNAi cells, and black bars indicate SIRT1-RNAi cells).

 Then, HepG2 cells were treated with the SIRT1 inhibitor Sirtinol O M) for 12 h to determine glycogen synthesis. The results are shown in Figure 2E. It can be seen that inhibition of SIRT1 by the SIRT1 inhibitor Sirtinol prevents insulin-induced glycogen synthesis.

 All of the above results indicate that a decrease in SIRT1 leads to a decrease in insulin sensitivity. Example 3 Up-regulation of the effect of SIRT1 protein levels on glucose uptake and insulin activation signals in normal and insulin resistance

 The virus HSV-SIRT1 was prepared according to the aforementioned method, and C2C12 cells were treated with HSV-SIRT1 to up-regulate SIRT1 protein levels in C2C12 cells, and their effects on glucose uptake and insulin activation signals under normal and insulin resistance were measured. The results are shown in Figures 3A-3C.

 As can be seen from Figure 3A, upregulation of SIRT1 protein levels has no significant effect on glucose uptake under normal conditions. In the figure, the white column indicates no insulin and HSV-SIRT1 group, the black column indicates single insulin group, the gray column indicates single plus HSV-SIRT1 group, and the black dot white column indicates insulin and HSV-SIRT1 group. .

 As can be seen from Figure 3B, up-regulation of SIRT1 protein levels significantly promoted glucose uptake in the case of insulin resistance. In the figure, the white column indicates no insulin and HSV-SIRT1 group, the black column indicates single insulin group, the gray column indicates single plus HSV-SIRT1 group, and the black dot white column indicates insulin and HSV-SIRT1 group. .

 Figure 3C is a graph showing the effect of SIRT1 protein levels on some insulin activation signals using conventional Western Blot. Western Blot detection of insulin signaling pathways revealed that overexpression of SIRT1 promotes insulin resistance, protein kinase B (Akt), glycogen synthase kinase 3p (Gsk3p), and phosphoinositide-dependent protein kinases in insulin resistance ( Phosphorylation levels of PDK1) (corresponding antibodies to the above insulin receptors or kinases were purchased from Cell Signal, Inc., USA). As can be seen from Figure 3C, up-regulation of SIRT1 protein levels enhances insulin activation signals.

 In summary, up-regulation of SIRT1 protein levels has no effect on glucose intake and insulin activation signals under normal conditions, but can significantly enhance glucose uptake and insulin activation signals in insulin resistance. Example 4 Effect of resveratrol treatment on glucose uptake and insulin activation signals

 Treatment of C2C12 cells with resveratrol (concentration 0.01, 0.1, Ι.Ο μΜ), thereby promoting in C2C12 cells

The expression or activity of SIRT1 was determined for its effect on glucose uptake and insulin activation signals in both normal and insulin resistance. The results are shown in Figure 4Α - Figure 4C.

As can be seen from Figure 4, resveratrol treatment promotes glucose uptake under normal conditions. In the figure, the white column indicates no insulin and resveratrol groups, the black column indicates the single plus insulin group, the gray column indicates the single plus resveratrol group, and the black dot white column indicates the addition of insulin and chalk. Resin group. As can be seen from Figure 4B, resveratrol treatment promotes glucose uptake in the case of insulin resistance. In the figure, the white column indicates no insulin and resveratrol groups, the black column indicates the single plus insulin group, the gray column indicates the single plus resveratrol group, and the black dot white column indicates the addition of insulin and chalk. Resin group.

 As can be seen from Figure 4C, resveratrol treatment enhanced insulin activation signaling and resveratrol significantly promoted SIRT1 protein expression. Western Blot detection of insulin signaling pathways showed that resveratrol treatment promotes insulin receptor, protein kinase B (Akt), glycogen synthase kinase 3β (Ο 3β) and phosphoric acid in normal and insulin resistance. Phosphorylation levels of alcohol-dependent protein kinase (PDK1) (corresponding antibodies to the above insulin receptors and individual kinases were purchased from Cell Signal, Inc., USA).

 In summary, Resveratrol treatment promotes glucose uptake and enhances insulin activation in both normal and insulin resistance. Example 5 Resveratrol and SIRT1 enhance hepatocyte glycogen synthesis

 The HepG2 cells infected with the lentivirus prepared as described above (i.e., Sirt RNAi virus) were subjected to RNA interference in the intracellular SIRT1, resulting in a decrease in SIRT1 protein expression. The control was to infect HepG2 cells with Luc RNAi virus (i.e., control virus).

 Cells were treated with insulin and resveratrol, respectively. The results are shown in Figure 5.

 It can be seen from the results that resveratrol enhances hepatocyte glycogen synthesis requiring the involvement of SIRT1. Example 6 Up-regulation of SIRT1 protein levels or effects of resveratrol treatment on PTP-1B

 According to the above method, HSV-SIRT1 was used to up-regulate SIRT1 protein level, Western Blot was used to determine the effect of PTP-1B protein CPTP-1B antibody purchased from Upstate, USA, using Tubulin as control; using the above materials and methods, part 6 The RT-PCR method described PTP-1B mRNA levels, with Actin as a control. The results are shown in Figures 6A-6B.

 As shown in Figure 6A, it was found that up-regulation of SIRT1 protein levels reduced PTP-1B protein levels in insulin resistance, and the higher the SIRT1 protein level, the lower the PTP-1B protein level.

 As shown in Figure 6B, it was found that up-regulation of SIRT1 protein levels reduced PTP-1B mRNA levels in the case of insulin resistance. Figure 6E is a quantification of the PTP-1B mRNA detection bands of Figure 6B.

 Next, the inventors also treated cells with resveratrol (concentrations of 0.01, 0.1, and 1.0 μM, respectively), and promoted the activity and expression level of SIRT1 in C2C12 cells, and the effect of PTP-1B protein by Western Blot was determined. Tubulin was used as a control; PTP-1B mRNA levels were determined using the RT-PCR method described in Section 6 of the aforementioned Materials and Methods. The results are shown in Figures 6C and 6D.

 As shown in Figure 6C, resveratrol treatment was found to significantly reduce PTP-1B protein levels in both normal and insulin resistance.

 As shown in Figure 6D, resveratrol treatment was found to significantly reduce PTP-1B mRNA levels in both normal and insulin resistance. Figure 6F is a quantification of the PTP-1B mRNA detection bands of Figure 6D.

In summary, up-regulation of SIRT1 protein levels (in the case of insulin resistance; or resveratrol treatment (normal or insulin resistance) can reduce PTP-1B protein and mRNA levels. Example 7 Up-regulation of PTP-1B protein levels on the up-regulation of SIRT1-induced increase in insulin sensitivity. According to the aforementioned method, HSRT-SIRT1 and HSV-PTP-1B were used to up-regulate SIRT1 protein levels and PTP-1B protein in C2C12 cells, respectively. Level.

 The HSV-mediated up-regulation of PTP-1B and SIRT1 protein levels as shown by the Western Blot method described above is shown in Figure 7A.

 As shown in Figure 7B, upregulation of PTP-1B protein reversed the increase in glucose uptake caused by up-regulation of SIRT1 protein levels. Upregulation of SIRT1 protein levels leads to an increase in glucose uptake without up-regulating PTP-1B protein levels, whereas up-regulation of SIRT1 protein levels leads to a gradual decrease in glucose uptake when PTP-1B protein levels are gradually upregulated. Among them, the white column indicates that insulin is not added and SIRT1 and PTP-1B are not up-regulated, the black column indicates that insulin is added and SIRT1 is up-regulated and PTP-1B is not up-regulated, and the gray column indicates that insulin is added and SIRT1 is up-regulated and PTP_1B is up-regulated.

 Therefore, it can be seen that up-regulation of PTP-1B protein level can reverse the increase in insulin sensitivity caused by up-regulation of SIRT1, indicating that the effect of SIRT1 found in the present invention to increase insulin sensitivity is achieved by inhibiting PTP-1B. Example 8 Effect of resveratrol on diabetes-related symptoms such as insulin sensitivity

 The insulin resistance model of C57/BL6 mice was induced by high fat as described above, and resveratrol (2.5 mg/kg/d) was administered while feeding high fat diet. Four weeks later, the glucose tolerance (GTT) and insulin tolerance (ITT) of the mice were measured by a blood glucose meter, and serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) levels were measured by an automatic biochemical analyzer. Serum insulin levels were determined by radioimmunoassay. The results are shown in Figures 8A-8E.

 As can be seen from Fig. 8A, in the low-fat group, the serum insulin level in the animal is low; in the high-fat group, the serum insulin level in the animal is high; and in the high-fat group, the serum insulin level is significantly lower after the resveratrol is administered in the high-fat group. In the high fat group. Therefore, it can be seen that resveratrol can inhibit hyperlipidemia induced by high fat.

 Hungry overnight mice were injected with a dose of glucose and then measured for blood glucose concentration (GTT) at different time points. As can be seen from Fig. 8B, the blood glucose level of the high fat group was significantly higher than that of the low fat group and the high fat ten resveratrol group in each time period. Therefore, resveratrol can improve high glucose-induced glucose tolerance.

 Hungry overnight mice were injected with a dose of insulin and then tested for blood glucose concentration (ITT) at different time points. As can be seen from Fig. 8C, the blood glucose level of the high fat group was significantly higher than that of the low fat group and the high fat ten resveratrol group in each time period. Therefore, resveratrol can improve insulin resistance induced by high fat.

 As can be seen from Fig. 8D, in the low-fat group, the total cholesterol content in the animal is low; in the high-fat group, the total cholesterol content in the animal is high; and in the high-fat group, the total cholesterol content is lower than that in the high-fat group. High fat group. Thus, it can be seen that resveratrol can reduce the increase in total cholesterol levels induced by high fat.

 It can be seen from Fig. 8 that in the low-fat group, the low-density lipoprotein content in the animal is low; in the high-fat group, the low-density lipoprotein content in the animal is high; and in the high-fat group, the low-density lipoprotein is low. The density lipoprotein content is lower than that of the high fat group. Therefore, it can be seen that resveratrol can reduce high-fat-induced elevation of low-density lipoprotein.

In conclusion, resveratrol can improve diabetes-related symptoms such as insulin sensitivity and reduce total cholesterol and low-density lipoprotein levels in mice with high-fat diet. Example 9 Method for screening for potential substances that increase insulin sensitivity method 1:

 Targeting the SIRT1 protein, muscle cells (such as C2C12 used in the present invention) or hepatocytes (such as HepG2 cells) and other types of cells (such as HEK293T cells) are used to screen for potential substances that increase insulin sensitivity.

 Test group: A candidate substance was added to the aforementioned insulin-responsive cells.

 Control group: No candidate substance was added to the aforementioned insulin-responsive cells.

 Detection of SIRT1 protein activity or expression in test group cells, and in control cells

 Comparison of SIRT1 protein activity or expression, if the SIRT1 protein activity or expression in the test group is statistically higher than (e.g., 20% higher or higher) in the control group, it indicates that the candidate is a substance that can be used to increase insulin sensitivity.

 details as follows:

 The luciferase reporter plasmid pSIRT1-luciferase transfected with the SIRT1 promoter was transfected with various Chinese herbal extracts (final concentration of lOO g/ml) using a reporter gene assay (see Nutrient Availability Regulates SIRT1 Through a Forkhead- Dependent Pathway, SCIENCE VOL 306, 17 DECEMBER 2004) and internal control plasmid β-gal (purchased from Promega, ie pSV-β-Galactosidase Control Vector) HEK293T cells (purchased from ATCC) 24 hours later, the determination of Chinese medicine for SIRT1 initiation Sub-control of the expression of luciferase, which reflects the effect on SIRT1 expression. Some of the results are shown in the figure. The horizontal axis represents the Chinese medicine number and c represents the control. The vertical axis represents the activity of luciferase relative to the control.

The reporter gene assay method was as follows: HEK293T cells were seeded at a density of 1×10 ⁴ cells per well in 96-well plates, and cultured at 37 ° C, 5% CO _{2 for} 24 hours, and the SIRT1 promoter was used to control the expression of the luciferase plasmid pSIRT1-luciferase. And the internal control plasmid β-gal was transferred to each well by calcium phosphate coprecipitation. After 16 hr, the supernatant was discarded, the cells were washed with PBS, and the cell culture medium was changed. At the same time, the test Chinese medicine was added to a final concentration of 100 μg/ml, and set. Corresponding negative control group. After gently mixing, incubate at 37 °C, 5% CO ₂ , discard the supernatant after 24 hours, wash the cells with PBS, add 100 l PLB cell lysate (Promega) per well, and collect the cell lysate with Promega fluorescence. The luciferase assay was performed on a prime enzyme reporter assay kit.

 The results are shown in Fig. 9. As can be seen from the figure, the extract of barley, numbered 110, the extract of Clematis, numbered 130, the extract of five skins, numbered 146, and the extract of Tripterygium wilfordii, numbered 161, No. 191, the extract of sedum, the rush extract of 210, the extract of Zhu Dengxin, number 211, the extract of capillaris, number 215, the black ugly extract number 218, the agarwood number 249 Extract, fried hexagram extract No. 270, rose flower extract No. 304, Inula flower extract number 365, Jade butterfly extract No. 388, Suzi extract No. 391, OB extract 405, oyster extract No. 412, Shichangpu extract No. 424, Radix Sophorae extract No. 428, Raw scutellaria extract No. 429, lock number 486 Yang extract, squid bone extract No. 502 was screened (all Chinese herbal extracts were purchased from Jiangyin Tianjiang Pharmaceutical Co., Ltd., which were routinely extracted, concentrated, dried, Granulated). It was found that the raw jaundice numbered 429 significantly increased the expression of SIRT1.

 In summary, using the SIRT1 protein as a target, a reporter gene or other appropriate method can be used to screen for potential substances that increase insulin sensitivity.

 Method 2:

Targeting PTP-1B, muscle cells that respond to insulin (such as C2C12 used in the present invention) or hepatocytes (such as HepG2 cells) are screened for potential substances that increase insulin sensitivity. Test group: A candidate substance was added to the aforementioned insulin-responsive cells.

 Control group: No candidate substance was added to the aforementioned insulin-responsive cells.

 The protein expression or mRNA level of PTP-1B in the test group cells was detected and compared with the protein expression or mRNA level of PTP-1B in the control group, if the protein expression or mRNA of PTP-1B in the test group was The level is statistically lower (e.g., 20% higher or higher;) in the control group, indicating that the candidate is a substance that can be used to increase insulin sensitivity. Example 10 Composition containing an appropriate amount of SIRT1 protein or an agonist or an upregulator thereof

 The formulation of the food composition is as shown in Table 1:

 Table 1

 The various functional foods described above were fed to C57/BL6 mice with high fat-induced insulin resistance, and the effects on insulin sensitivity in mice were tested. After a certain period of time, the mice were tested and found to be significantly improved for high-fat-induced hyperinsulinemia, high-fat-induced glucose tolerance, and the like. Example 11 Pharmaceutical Composition

 The pharmaceutical composition is formulated as shown in Table 2:

 Table 2

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

 A use of a SIRT1 protein or an agonist or an upregulator thereof for the preparation of a composition for improving insulin sensitivity.

 The use according to claim 1, wherein the SIRT1 protein or an agonist or an upregulator thereof is further used for the preparation of a composition which inhibits transcription or expression of protein tyrosine phosphatase-1B.

 3. The use according to claim 1, wherein the upregulator of the SIRT1 protein is a substance that increases the expression of SIRT1 protein.

 The use according to claim 1, wherein the composition is for treating a disease associated with a decrease in insulin sensitivity.

 The use according to claim 4, wherein the diseases associated with decreased insulin sensitivity include: insulin resistance, type 2 diabetes, hyperinsulinemia, diabetic ketoacidosis, hyperosmolar non-ketone Diabetes coma, lactic acidosis.

 6. The use according to claim 1, wherein the agonist or upregulator of the SIRT 1 protein is selected from the group consisting of: resveratrol, riviera, isoglycyrrhizin, quercetin, tetrahydroxyl芪, or Chinese medicine jaundice.

 7. A method of screening for potential substances useful for increasing insulin sensitivity, characterized in that the method comprises:

 The candidate substance is contacted with a system expressing the SIRT1 protein to detect the effect of the candidate substance on the SIRT 1 protein; if the candidate substance can increase the expression of the SIRT1 protein or promote the activity of the SIRT1 protein, it indicates that the candidate substance can be used to improve insulin sensitivity. Sexual potential substance.

 8. The method according to claim 7, further comprising:

 Observing the expression or activity of protein tyrosine phosphatase-1B in the system,

 If the expression or activity of protein tyrosine phosphatase-1B is decreased, it indicates that the candidate substance is a potential substance that can be used to increase insulin sensitivity.

 9. A substance obtainable by the method of claim 7 for improving insulin sensitivity.

 10. A composition, characterized in that said composition comprises:

 (i) an effective amount of a SIRT1 protein or an agonist or a modulator thereof;

 (ii) an effective amount of a substance selected from the group consisting of: biguanide diabetes drugs, sulfonylurea diabetes drugs, glucosidase inhibitor drugs, insulin sensitizing drugs, aldose reductase inhibitor drugs, insulinotropic drugs Release of drugs; and

 (iii) a pharmaceutically or food acceptable carrier.

 1 1. The composition according to claim 10, wherein the composition is a unit dosage form containing resveratrol 0.00001-0.01 g.

 12. A method of increasing insulin sensitivity in a patient, the method comprising: administering to the patient 0.00001-0.01 g/day of SIRT 1 protein or an agonist or an upregulator thereof.