CN101780268B - Method and reagent for diagnosing, preventing and treating insulin resistance - Google Patents

Abstract

The invention relates to a method and a reagent for diagnosing, preventing and treating insulin resistance. A method for diagnosing insulin resistance comprises the steps of detecting the expression level of beta arrestin 2 in a sample to be detected, and comparing the expression level of the beta arrestin 2 of the sample with that of a normal sample to diagnose the insulin resisting level, wherein relatively lower expression level of beta arrestin 2 indicates the generation of insulin resistance. The invention also discloses a reagent for preventing and treating insulin resistance and relative diseases.

Description

Be used for diagnosis, method and the reagent of prevention and treatment insulin resistant

Technical field

This invention treatment with insulin resistant widely is relevant.Say that more accurately it relates to β arrestin2 albumen and role in insulin sensitivity.This invention also relates to method and the reagent of diagnosis, prevention and treatment insulin resistant relevant disease.

Background technology

Insulin resistant (insulin insensitivity) became one of the most serious threat of public health in recent years.It causes the imbalance of body glucose dynamic equilibrium and with glucose intolerance, obesity, disorders of lipid metabolism, hypertension and cardiovascular disease close contacting is arranged.After insulin resistant took place, body directly caused hyperinsulinism symptom in the blood circulation for a large amount of insulin of the improper generation of decline of compensation insulin sensitivity.The final result of insulin resistant development is the generation of diabetes.

Insulin resistant refers to insulin in the blood circulation for fat, liver, the decline of glycolipid metabolism ability of regulation and control in the muscle.The reason of its generation is that Insulin receptor INSR is to the decline of insulin stimulating response.Usually under the state, insulin in the blood circulation is by fat, liver, the Insulin receptor INSR identification on muscle cell surface, phosphorylation IRS albumen (IRS proteins) after Insulin receptor INSR is activated, the phosphorylation of IRS albumen further mediates the activation of downstream PI3K signal path.The PI3K signal path has mediated the main metabolic function of insulin.

Insulin signaling pathway is a complexity and the signal network that is subjected to the height accuracy controlling.In this path, PI3K energy phosphorylation PI4,5-P ₂Produce the film anchoring molecule PI3 of Akt and PDKs, 4,5-P ₃In a single day Akt moves on the film, will and activate by the PDKs phosphorylation.The Akt of activation is kinases and the transcription factor in phosphorylation downstream successively, thereby mediates most insulin metabolism activity.

Defective such as key proteins such as Akt in the insulin signaling pathway all will cause severe insulin resistance.Existing bibliographical information Akt gene knockout (Cho, H.et al., " Insulin resistance and a diabetes mellitus-like syndrome inmice lacking the protein kinase Akt2 (PKB beta); " Science, 292,1728-31,2001) or the forfeiture (Takata of kinase activity, M.et al., " Requirement for Akt (protein kinase B) in insulin-induced activationof glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1), " J.Biol.Chem., 274,20611-8,1999) all directly cause insulin resistant.Even some are to active albumen such as the PP2A that plays regulating and controlling effect of Akt, the imbalance of TRB3 also can influence insulin resistant, thereby shown the pivotal role (Resjo of Akt in the insulin signaling transduction, S.et al., " Protein phosphatase 2A is the main phosphatase involved in the regulation ofprotein kinase B in rat adipocytes, " Cell Signal, 14,231-8,2002; And Du, K.et al., " TRB3:a tribbles homolog that inhibits Akt/PKB activation by insulin in liver, " Science, 300,1574-7,2003).

In view of the appearance of insulin resistant usually early than organism metabolic disorder and disease (as type-II diabetes, glucose do not tolerate, obesity, disorders of lipid metabolism, hypertension, cardiovascular disease, metabolic arthritis disease and Hyperinsulinism etc.) appearance, therefore find and the biomarker of definite insulin resistant will provide the new method of a kind of effective prevention and treatment insulin resistant and relevant disease thereof.

Summary of the invention

The expression of reconstruction β arrestin2 and active in prevention or treatment insulin resistant and relevant disease such as type-II diabetes, the forfeiture of glucose tolerance, obesity, disorders of lipid metabolism, hypertension, metabolic arthritis disease, Hyperinsulinism and cardiovascular disease etc. is useful.

In a first aspect of the present invention, provide a kind of β arrestin2 albumen or the purposes of adjustment on it (as improving the reagent (up-regulated agent) of β arrestin2 expression in the destination organization), for the preparation of the compositions of prevention or treatment mammal insulin resistant or insulin resistant relevant disease.

In another preference, described insulin resistant or insulin resistant relevant disease are selected from: type-II diabetes, hypertension, disorders of lipid metabolism, cardiovascular disease, obesity, Hyperinsulinism, metabolic arthritis disease, or glucose intolerance.

In another preference, the upward adjustment of described β arrestin2 albumen comprises the expression vector of the polynucleotide of coding β arrestin2.

In another preference, described expression vector is plasmid vector.

In another preference, described expression vector is viral vector.

In another preference, described viral vector is selected from down group: retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.

In another preference, described mammal is the people.

In another aspect of this invention, provide a kind of purposes of β arrestin2 albumen, for the preparation of the reagent of diagnosis insulin resistant or insulin resistant relevant disease.

In another aspect of this invention, provide the reagent purposes of a kind of specific recognition β arrestin2 albumen or its encoding gene or transcript, for the preparation of the test kit of diagnosis insulin resistant or insulin resistant relevant disease.

In another preference, the reagent of described specific recognition β arrestin2 albumen or its encoding gene or transcript is selected from: the antibody of the anti-β arrestin2 of specificity; Or the primer of specific amplification β arrestin2mRNA.

In another aspect of this invention, provide a kind of method of diagnosing insulin resistant, comprising:

Analyze the expression of β arrestin2 in the testing sample, and with normal specimens in β arrestin2 expression compare, the β arrestin2 expression in the testing sample is relatively low to indicate that object to be measured has insulin resistant.

In another preference, the expression of β arrestin2 is its protein expression level of antibody test that utilizes β arrestin2 in the described analysis testing sample.

In another preference, the expression of β arrestin2 is to utilize the reverse transcriptional PCR primer to detect its mRNA expression in the described analysis testing sample.

In another aspect of this invention, provide the method for a kind of prevention or treatment insulin resistant or insulin resistant relevant disease, comprise the reagent that can improve β arrestin2 expression in the destination organization that gives mammal treatment effective dose.

In another aspect of this invention, provide a kind of diagnostic kit of diagnosing insulin resistant or insulin resistant relevant disease, this test kit comprises: the reagent of specific recognition β arrestin2 albumen or its encoding gene or transcript.

In another preference, the reagent of described specific recognition β arrestin2 albumen or its encoding gene or transcript is selected from: the antibody of the anti-β arrestin2 of specificity albumen, or specific amplification β arrestin2mRNA's is a pair of or several to the reverse transcriptional PCR primer.

In another aspect of this invention, the purposes that a kind of β arrestin2 albumen is provided or adjusts on it is for the preparation of the compositions that promotes that Insulin receptor INSR (IR)/Akt/ β arrestin2/Src signal transduction complex forms.

In another preference, a kind of signal transduction complex (being preferably the complex of separation) is provided, described complex comprises: Insulin receptor INSR, Akt, β arrestin2 and Src.

In another preference, the purposes of described signal transduction complex is provided, be used for the material of screening prevention or treatment mammal insulin resistant or insulin resistant relevant disease.The present invention proposes a kind of new method of diagnosis of insulin resistant.Analyze the expression of the β arrestin2 of testing sample, and comparison β arrestin2 expression in testing sample and normal specimens is diagnosed its insulin resistant level: the generation that relatively low β arrestin2 expression is indicating its insulin resistant.

Another aspect of the present invention relates to the new method of a kind of prevention and treatment insulin resistant and relevant disease thereof.In one embodiment of the invention, this method comprises the reagent that can increase β arrestin2 expression in the particular organization that gives mammal treatment effective dose.

Another aspect of the present invention relates to the diagnostic kit of diagnosing insulin resistant.In one embodiment of the invention, this test kit comprises a kind of antibody of the β of detection arrestin2 albumen or the RT-PCR primer of one or several amplification β arrestin2mRNA.

To be set forth in the following description and claims about the more aspect of the present invention and advantage.

Description of drawings

Fig. 1 has shown the remarkable downward modulation of β arrestin2 (β arr2) in type-II diabetes model mice sample.A, in diabetic mice db/db mice (n=5) and normal mouse (n=5), β arrestin2 is at fat, liver, the protein expression level in the muscle.Shown the densitometry analysis result among the figure.B, in diabetic mice db/db mice (n=5) and normal mouse (n=5), β arrestin2 is at fat, liver, the mRNA expression in the muscle.The normal mouse that 8 ages in week, normal diet are fed, body weight is 20.3 ± 1.2g, food intake dose is that the db/db mice body weight that 3.5 ± 0.5g 8 ages in week, normal diet are fed is 38.5 ± 1.4g, food intake dose is 4.8 ± 0.7g.C, normal C57BL/6 mice (body weight 22.6 ± 2.1g in the normal diet nursing, food intake dose is the normal C57BL/6 mice (body weight 36.7 ± 1.9g of 3.6 ± 0.3g) (n=5) and high fat feeding (HFD), food intake dose is the fat of 4.1 ± 0.2g) (n=5), liver, the protein expression level of β arrestin2 in the muscle.Shown the densitometry analysis result among the figure.D, the normal C57BL/6 mice of feeding at normal diet (n=5) and high fat feeding (HFD) be the fat of C57BL/6 mice (n=5) normally, liver, the mRNA expression of β arrestin2 in the muscle.C57BL/6 mice reuse normal diet or the high lipid food in 6 ages in week fed for 8 weeks.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Fig. 2 has shown that the disappearance of β arrestin2 causes insulin resistant.β arrestin2 disappearance mice (n=8) and normal mouse (n=8) serum insulin levels (A) after taking food on an empty stomach and again, blood sugar level (B).β arrestin2 crosses expression mice (n=8) and the serum insulin levels (C) of normal mouse (n=8) after taking food on an empty stomach and again, blood sugar level (D).Glucose sugar resistance test (the GTTs) (1gkg of β arrestin2 disappearance mice (n=10) and normal mouse (n=8) ^-1) (E) insulin resistant test (ITTs) (1Ukg ^-1) (F).The AUC blood sugar level is presented at G.β arrestin2 crosses the GTTs (1gkg of expression mice (n=11) and normal mouse (n=8) ^-1) (H) ITTs (0.75Ukg ^-1) (I).The AUC blood sugar level is presented at J.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Fig. 3 has shown that applying β arrestin2 can recover insulin sensitivity.The β arrestin2 disappearance mice (n=8) in high 8 weeks of fat feeding and the GTTs (1.5gkg of normal mouse (n=8) ^-1) (A), ITTs (1.5Ukg ^-1) (B).The AUC blood sugar level is presented at C.The β arrestin2 in high 8 weeks of fat feeding crosses the glucose sugar tolerance GTTs (1.5gkg of expression mice (n=8) and normal mouse (n=8) ^-1), (D) insulin resistant experiment ITTs (1.5Ukg ^-1) (E).The AUC blood sugar level is presented at F.G, diabetic mice db/db mice (n=8) comprises the blood sugar level behind the adenovirus of β Gal or β arrestin2 in injection.Diabetic mice db/db mice (n=8) comprises the glucose sugar tolerance GTTs (1.5gkg of adenovirus after 7 days of β Gal or β arrestin2 in injection ^-1) (H), insulin resistant experiment ITTs (1.5Ukg ^-1) (I) and AUC blood sugar level (J).The mice that high fat is induced, the mice high lipid food in 6 ages in week fed for 8 weeks again, and the db/db mice is accepted the adenovirus injection when 8 ages in week.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Fig. 4 has shown that the Akt that β arrestin2 can promote the Src of insulin stimulating to rely on activates.A, β arrestin2 lacks mice (n=6), and β arrestin2 crosses insulin activation PI3K in expression mice (n=6) and normal mouse (n=6) liver.Mice to be measured is with saline or insulin (1U/kg ^-1) injected 10 minutes, immune purification PI3K and measure its active B with ELISA from liver extract, C, β arrestin2 disappearance mice (n=6) (B), β arrestin2 cross expression mice (n=6) (C) and normal mouse (n=6) liver in Akt and downstream substrate thereof be subjected to the activation of insulin.The preparation of orgotein extract is used for being pAkt with reference to A, the immunoassay of pFoxo1 and pGSK3.D, diabetic mice db/db mice (n=8) the adenovirus of injection β Gal or β arrestin2 after 7 days in the liver Akt and downstream substrate thereof activated by insulin.E, the Akt that Src relies on activates.The Hep3B hepatocyte need not or be handled 30 minutes with 10 μ M Src inhibitor PP2 or 100nM PI3K inhibitor Wortmanin, used the 100nM insulin stimulating then, had shown the phosphorylation level (n=6) of Akt at serine/threonine or tyrosine among the figure.F, β arrestin2 lacks mice (n=6), and β arrestin2 crosses the phosphorylation level of Akt tyrosine insulin-induced in expression mice (n=6) and normal mouse (n=6) liver.Akt obtains with immune purification in the protein extract of above-mentioned B and C, prints and dyes to measure with immunity with the antibody of tyrosine of antagonism phosphorylation again.Testing used mice all was 8 ages in week, and normal diet is fed.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Fig. 5 has shown that β arrestin2 can mediate the interaction of Akt/Sr.A, immune precipitation determination the interaction (n=5) of Akt/ β arrestin2/Src in the C57BL/6 mouse liver lysate.B, Akt and Src can interact in the brood mouse liver of wild type, but the two can not interact in β arrestin2 disappearance mice (n=6).The orgotein extract of these mices is precipitated by the antibody mediated immunity of Anti-Akt and Anti-Src.The interaction that C, β arrestin2 cross Akt/Src in expression mice (n=6) liver strengthens.The orgotein extract that β arrestin2 crosses expression mice (n=6) and the brood mice of wild type (n=6) is precipitated by the antibody mediated immunity of Anti-Akt and Anti-Src.D, the interaction characteristic of pictorialization β arrestin2 and truncate mutation body thereof and Akt and Src.E, β arrestin2 1-185 and Src interact but not with Akt interact (n=3).Cross expression Akt or Src and expression or do not express HA-β arrestin2, the HA-β arrestin2 in the Hep3B cell of HA-β arrestin2 1-185 is earlier with after the immunoprecipitation, and reuse anti-HA antibody mediated immunity trace detects.F crosses the Hep3B cell of expressing HA-β arrestin21-185 or β Gal and precipitates with the anti-Akt antibody mediated immunity, and the Akt in the precipitation and Src detect (n=3) with immunoblotting.G, the Akt that β arrestin2 1-185 inhibition is insulin-induced and the activation of downstream substrate thereof.To crossing the Hep3B hepatocyte of expressing HA-β arrestin2 1-185 or β Gal, earlier with insulin stimulating, analyze pAkt with immunoblotting again, pFoxo1, and pGSK3.Testing used mice all is that 8 ages in week, normal diet are fed.

Fig. 6 shows the formation of insulin stimulating Receptor/Akt/ β arrestin2/Src signal complex.A, the formation of insulin-induced Akt/ β arrestin2/Src signal complex.C57BL/6 mice (n=6) is injected the insulin of 1U/kg, prepare its orgotein extract and be used for immunoprecipitation, the antibody of used immunoprecipitation is Anti-Akt antibody, and immunoblotting detects Akt, β arrestin2 and the Src albumen in the immunoprecipitation complex.B, the interaction of insulin stimulating Akt/ β arrestin2/Src signal complex and Insulin receptor INSR.The orgotein extract is according to the method preparation of describing among the A, and the extract of preparation is used for immunoprecipitation IR β, and immunoblotting detects and the interactional Akt of IR β, β arrestin2 and Src (N=6).C, β arrestin2 is necessary for the interaction of Akt/Src and IR.The orgotein extract of crossing preparation expression mice (n=6) and the brood mice of their wild type (n=6) from β arrestin2 disappearance mice (n=6), β arrestin2 is used for immunoprecipitation IR β.Immunoblotting detects and the interactional Akt of IR β and Src.D, β arrestin2186-409 and IR β interact but do not interact with Akt and Src.Overexpression HA-β arrestin2, the Hep3B hepatocyte of HA-β arrestin2186-409 or β Gal is cleaved, and with corresponding albumen in the antibody precipitation lysate separately, immunoblotting detects the HA-tagged β arrestin (n=3) in the precipitate.E, the Akt that β arrestin2186-409 inhibition is insulin-induced and the activation of downstream substrate thereof.The Hep3B hepatocyte of overexpression β arrestin2186-409 or β Gal is cleaved, and lysate is used for immunoblotting assay pAkt, pFoxo1 and pGSK3.Used mice all is the mice in 8 ages in week of normal diet nursing in this experiment.

Fig. 7 shows that the expression of β arrestin2 1-185 or 186-409 mutant causes insulin resistant.A, C57BL/6 mice (n=8) comprises the blood sugar level behind the adenovirus of β Gal or β arrestin2 1-185 or 186-409 in injection.C57BL/6 mice (n=8) comprises the glucose sugar tolerance GTTs (1gkg behind the adenovirus of β Gal or β arrestin2 1-185 or 186-409 in injection ^-1) (B), ITTs (1Ukg ^-1) (C).The AUC blood sugar level is presented at D.The C57BL/6 mice was 8 ages in week, and normal diet is fed.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.E, the sketch map of the formation of insulin stimulating Receptor/Akt/ β arrestin2/Src signal complex.

Fig. 8 has shown the downward modulation of β arrestin1/2 in the type-II diabetes patient samples.A, in the diabetic sample, the protein expression level of β arrestin1/2 (on).Sample matched according to the age.Down: the statistics (n=8) of β arrestin1/2 protein expression level.B, the patient's who studies among the A clinical, biochemical characteristics.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Fig. 9 shows that β arrestin2 promotes the insulin sensitivity of liver and muscle.A, B, β arrestin2 lacks mice (n=10), and β arrestin2 crosses expression mice (n=10) and body weight and the dietary amount of the brood mice of their wild type (n=10) under the normal diet condition, and the expression of β arrestin1/2.C, β arrestin2 lacks mice (n=10), and β arrestin2 crosses expression mice (n=11) and the brood mice of their wild type (n=8) GTTs (1g/kg ^-1) insulin level in the process.D, E, high fat diet β arrestin2 lacks mice (n=8), and β arrestin2 crosses body weight and the dietary amount of expression mice (n=8) and the brood mice of their wild type (n=8).F, G, injection contains body weight and the dietary amount of diabetes db/db mice under the normal diet condition of the adenovirus of β Gal (n=8) or β arrestin2 (n=8), and the expression of β arrestin1/2.H, β arrestin2 lacks mice (n=6), and β arrestin2 crosses the white adipose tissue weight under expression mice (n=6) and the brood mice of their wild type (n=6) the normal diet condition.I, β arrestin2 lacks mice (n=6), and β arrestin2 crosses the white adipose tissue fat cell size (H/E dyeing) under expression mice (n=6) and the brood mice of their wild type (n=6) the normal diet condition.The β arrestin2 disappearance mice (n=6) that J, normal diet feed, β arrestin2 crosses expression mice (n=6) and the brood mice of their wild type (n=6)) free fatty acid (FFA) under starvation, triglyceride and cholesterol levels.K, the β arrestin2 disappearance mice (n=6) that normal diet is fed, β arrestin2 crosses expression mice (n=5) and the brood mice of their wild type (n=5) expression of G6p and pepck mRNA in the liver under starvation, and data are to be obtained by the quantitative RT-PCR analysis.L, the β arrestin2 disappearance mice (n=6) that normal diet is fed, β arrestin2 crosses expression mice (n=6) and the former foster hepatocyte of being commissioned to train of the brood mice of their wild type (n=6) is subjected to or is not subjected to the glucose output level of 10mM insulin processing after 3 hours.M, from the β arrestin2 disappearance mice (n=6) that normal diet is fed, β arrestin2 crosses the Ex vivo glucose of the musculus soleus that separates in expression mice (n=6) and their the brood mice of wild type (n=6) and takes in.Testing used mice all is all to be 8 ages in week.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Figure 10 shows that β arrestin2 strengthens the insulin signaling of liver and muscle.A, the vitro kinase experiment detects β arrestin2 disappearance mice (n=4), and β arrestin2 crosses the activity of Akt in expression mice (n=4) and normal mouse (n=4) liver.The mice fasting in 8 ages in week that normal diet is fed is pump pickle or insulin 10 minutes after 6 hours, and its orgotein extract is used for immunoprecipitation Akt, carries out the vitro kinase experiment.B, Fig. 4 B, the densitometry analysis of 4C.C, the densitometry analysis of Fig. 4 D.D, the densitometry analysis of Fig. 4 F.E, Akt Tyr315/326Ala show active the reduction under insulin stimulating.Express flag-Akt or flag-Akt 2M (Tyr315/326Ala) Hep3B hepatocyte extract with Akt or the Akt 2M of anti-flag antibody mediated immunity precipitation band flag, use anti-pTyr, anti-pAkt (ser473) and anti-flag antibody test (n=3).F, β arrestin2 lacks mice (n=6), and β arrestin2 crosses the activation of the Akt of insulin stimulating in expression mice (n=6) and the brood mice of their wild type (n=6) muscle.Pump pickle or insulin 10 minutes 8 ages in week mice to be measured the muscle protein extract be used for carrying out the total Akt of immunoblotting assay and activated Akt (p-Thr308, p-Ser473).G, the densitometry analysis that has active Akt level among the F.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Figure 11 shows that β arrestin2 mediation Akt/Src interacts.A, the densitometry analysis of Fig. 5 B.B, the densitometry analysis of Fig. 5 C.C, the combination of β arrestin2 120-409 and Src and Akt (n=4) data are expressed as mean+/-standard error in the Hep3B hepatocyte, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Figure 12 shows the formation of insulin stimulating Receptor/Akt/ β arrestin2/Src signal complex.A, external PULL DOWN test has confirmed the direct interaction of β arrestin2 and IR.In vitro transcription, translation [ ³⁵S] β arrestin2 and from the Hep3B hepatocyte IR of immunoprecipitation be used for external PULL DOWN test.B-C, the densitometry analysis of Fig. 6 A-B.D, Wortmannin handle the Akt/Src interaction (n=3) that suppresses β arrestin2 mediation.The Hep3B hepatocyte is used or handled 30 minutes without 100nM Wortmanin, uses the 100nM insulin stimulating then.With anti-IR or anti-Src antibody mediated immunity precipitation, detect with the specific antibodies shown in the figure then.E, the densitometry analysis of Fig. 6 C.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

Figure 13 shows that the expression of β arrestin2 1-185or 186-409 mutant increases the weight of the insulin resistant of db/db mice.A, db/db mice (n=8) is the blood sugar level in the specified number of days after injection comprises the adenovirus of β Gal or β arrestin2 1-185 or 186-409.Db/db mice (n=8) comprises the glucose of measuring in the 7th day sugar tolerance GTTs (1gkg behind the adenovirus of β Gal or β arrestin21-185 or 186-409 in injection ^-1) (B) insulin resistant experiment ITTs (1Ukg ^-1) (C).The AUC blood sugar level is presented at D.Testing used is that the db/db mice is that 8 ages in week, normal diet are fed.Data are expressed as mean+/-standard error, and result of the test difference table compared with the control is shown ^*P＜0.05, ^*P＜0.005.

The specific embodiment

Some embodiments of this invention relate to the diagnosis for insulin resistant, prevention and treatment useful reagent and method.In view of the appearance of insulin resistant usually early than the appearance of organism metabolic disorder and disease, can predict that therefore prevention also control insulin resistant will be conducive to the development of its relevant disease of prevention as early as possible.This invention has found that based on the inventor β arrestin2 plays an important role in insulin signaling pathway.

Arrestins is the protein family of the signal path of g protein coupled receptor (GPCRs) mediation in the important adjusting cell.GPCRs constitutes the family of the maximum of cell receptor, participates in cell function widely.Arrestins participates in the activity of GPCRs and regulates.

After agonist was in conjunction with GPCRs, GPCRs was activated and recruits trimerical G albumen and transmit signal.In case these receptors are activated, the stimulation that they also need to be switched off or to be adjusted to continuing has suitable reaction, and for example, the receptor that is activated needs by reticent.Reticent mechanism relates to by the receptor kinase of G albumen coupling (GRKs) phosphorylation.This phosphorylation labelling makes activated receptors be combined with Arrestin.In case Arrestins is attached to activated receptors, these receptors just can not the repeated transmission delivery signal.Thereby Arrestins plays an important role in the GPCRs of agonist mediation desensitization.

Except can ending the signal transduction of GPCRs, Arrestins also is found to participate in the specific function of receptor (non-GPCRs) of other types.In these other receptor signal transductions, Arrestins is mainly as the function of scaffolding protein.β arrestins and receptor form complex, β arrestins is by the joint albumen as the signal transduction complex in these complex, receptor and diversified signal path or effect protein are linked together, thus determined that signaling molecule is located in born of the same parents, transposition in the born of the same parents, self stability or with the characteristic of aspects such as other signaling molecule interaction.

For example, at β ₂After AR or Neurokinin 1 (NK-1) receptor are activated, β arrestins will with the activated receptors combination, and the kinases (ERK1/2) of recruiting Src and downstream thereof forms the signal complex on activated receptors.Referring to, DeFea, K.A.et al., " The proliferative and antiapoptotic effects of substance P arefacilitated by formation of a beta-arrestin-dependent scaffolding complex; " Proc.Natl.Acad.Sci.U.S.A., 97,11086-91,2000; And Luttrell, L.M.et al., " Beta-arrestin-dependentformation of beta2 adrenergic receptor-Src protein kinase complexes, " Science, 283,655-61,1999.Equally, comprise ERK path (e.g., Raf what angiotensin II type 1A receptor (AT1aR) stimulate to form, MEK and ERK) and JNK path (e.g., ASK1, MKK4 and JNK3) the signal complex in, β arrestins plays the effect of scaffolding protein.Referring to, Luttrell, L.M.et al., " Activation and targetingof extracellular signal-regulated kinases by beta-arrestin scaffolds, " Proc.Nat.l Acad.Sci.U.S.A., 98,2449-54,2001; And McDonald, P.H.et al., " Beta-arrestin 2:a receptor-regulatedMAPK scaffold for the activation of JNK3, " Science, 290,1574-7,2000.The signal complex of discovering these β arrestins mediations is essential for the transduction of receptor signal and the normal physiological function in downstream.

Although the combination of β arrestins and non-GPCRs is found, also there is not the combination of evidence proof this non-GPCRs of IR and β arrestins.The one side of this invention just be based on β arrestin2 albumen not only can in conjunction with IR and in the Insulin receptor INSR signal path as this new discovery of irreplaceable scaffolding protein.More specifically, the inventor finds that β arrestin2 is subjected to Akt and Src to be connected to IR after the stimulation of insulin.In addition, the inventor also find the correct formation of IR/Akt/ β arrestin2/Src signal complex be insulin signaling transduction with and the downstream metabolic function be indispensable.

Such as, the inventor finds that the downward modulation of β arrestin2 expression or the sudden change of function can both hinder the insulin signaling transduction and worsen insulin resistant.The inventor finds that also the signal transduction complex that relies on β arrestin2 participates in this insulin signaling transduction.In this complex, the generation that has caused insulin resistant unusually of β arrestin2 function and the development of type-II diabetes.These discoveries have pointed out β arrestin2 can become diagnosis and control insulin resistant and relevant disease such as type-II diabetes, the forfeiture of glucose tolerance, obesity, disorders of lipid metabolism, hypertension, metabolic arthritis disease, the new action target spot of Hyperinsulinism and cardiovascular disease etc.Hereinafter this discovery is described in detail.

In the present invention, used β arrestin2 albumen can be naturally occurring, such as its can be separated or purification from mammal.In addition, described β arrestin2 albumen also can be artificial preparation, such as producing recombinant beta arrestin2 albumen according to the genetic engineering recombinant technique of routine.Preferably, the present invention can adopt the β arrestin2 albumen of reorganization.

Any suitable β arrestin2 albumen all can be used for the present invention.Described β arrestin2 albumen comprises β arrestin2 albumen or its bioactive fragment (or being called active fragment) of total length.For example, the aminoacid sequence of described β arrestin2 albumen can with the GenBank accession number: the sequence shown in the NM145429 is substantially the same.

The aminoacid sequence of the β arrestin2 albumen that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included among the present invention.β arrestin2 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and described sequence of replacing through aminoacid does not influence its activity or kept the activity of its part.Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented at an easy rate, and guarantees not change the biological activity of gained molecule.These technology are recognized those skilled in the art, in general, can not change biological activity basically at the inessential area change single amino acids of a peptide species.See Molecular Biology of TheGene such as Watson, the 4th edition, 1987, The Benjamin/Cummings Pub.Co.P224.

The bioactive fragment of any β arrestin2 albumen can be applied among the present invention.Here, the implication of the bioactive fragment of β arrestin2 albumen refers to that as a peptide species it still can keep all or part of function of the β arrestin2 albumen of total length.Generally, described bioactive fragment keeps the activity of 50% total length β arrestin2 albumen at least.Under preferred condition, described active fragment can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length β arrestin2 albumen.

The present invention also can adopt β arrestin2 albumen modified or improvement, such as, can adopt the β arrestin2 albumen of being modified or improveing for the effectiveness that promotes its half-life, effectiveness, metabolism and/or albumen.Described can be to have less common ground with naturally occurring β arrestin2 albumen through the β arrestin2 albumen of modifying or improve, but also can prevent or treat mammiferous insulin resistant, and can not bring other harmful effect or toxicity.That is to say that any bioactive version that does not influence β arrestin2 albumen all can be used among the present invention.

The present invention has also comprised the nucleic acid of separation of the bioactive fragment of the described β arrestin2 albumen of encoding, and also can be its complementary strand.The DNA sequence of the bioactive fragment of coding β arrestin2 albumen can the complete sequence synthetic, and also the method for available pcr amplification obtains.After the DNA sequence of the bioactive fragment of the described β arrestin2 albumen that obtained to encode, it is connected into suitable expression vector, change proper host cell again over to.By cultivating the host cell after transforming, obtain desired albumen by separation and purification at last.

The present invention has also comprised the carrier of the nucleic acid molecules of the bioactive fragment that comprises the described β arrestin2 albumen of encoding.Described carrier also can comprise the expression regulation sequence that links to each other with the series of operations of described nucleic acid molecules, so that the expression of albumen.Described " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and namely the activity of same linear DNA sequence other parts can be regulated or control to some part of linear DNA sequence.For example, if the transcribing of promoter control sequence, it is exactly operationally to be connected in coded sequence so.Described expression vector for example is a viroid carrier.Described viral vector can be selected from down group: retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.Preferable viral vector is adenovirus vector.

The present invention also comprises the upward adjustment of β arrestin2 albumen, and it can be by raising expression or the activity of β arrestin2 albumen, and performance prevents or treat the effect of insulin resistant or insulin resistant relevant disease.As used herein, described upward the adjustment comprised agonist, promoter etc.The material of transcribing and translating of the activity of any β of raising arrestin2 albumen, the stability of keeping β arrestin2 albumen, the expression that promotes β arrestin2 albumen, prolongation β arrestin2 albumen effective acting time or promotion β arrestin2 all can be used for the present invention, as the material that can be used for preventing or treating insulin resistant or insulin resistant relevant disease.

Based on the inventor's above-mentioned new discovery, can also be with the mark of β arrestin2 as diagnosis insulin resistant or insulin resistant relevant disease: (i) carry out typing, Differential Diagnosis and/or the susceptibility analysis of insulin resistant or insulin resistant relevant disease; The (ii) relevant crowd's of assessment insulin resistant or insulin resistant treating correlative diseases medicine, curative effect of medication, prognosis, and select suitable Therapeutic Method; (iii) assess the ill risk of relevant crowd's insulin resistant or insulin resistant relevant disease in early days, the early monitoring early prevention and treatment.Such as, the separable crowd who goes out to cause insulin resistant or insulin resistant relevant disease by β arrestin2 abnormal gene expression, thus can treat more targetedly.

Therefore, can utilize β arrestin2 albumen to prepare the reagent of diagnosis insulin resistant or insulin resistant relevant disease, described reagent for example is specific recognition or in conjunction with the reagent of β arrestin2 albumen, as utilize a series of antibody of β arrestin2 protein Preparation, from these antibody, find the good antibody that can be used for clinical diagnosis of binding specificity.

Therefore, the present invention also provides the purposes of the reagent of a kind of specific recognition β arrestin2 albumen or its encoding gene or transcript, for the preparation of the test kit of diagnosis insulin resistant or insulin resistant relevant disease.

Whether and expression can adopt existence that various technology known in the art detect β arrestin2 gene or its transcript, these technology all comprise in the present invention.For example available existing technology such as Southern blotting, Western blotting, dna sequence analysis, PCR etc., these methods can be used in combination.

Whether the present invention also provides the existence that is used for detecting β arrestin2 gene at analyte and the reagent of expression.Preferably, when carrying out the detection of gene level, can adopt the primer of specific amplification β arrestin2; Or the existence that the probe of specific recognition β arrestin2 is determined β arrestin2 gene whether; When carrying out the detection of protein level, the expression that can adopt specificity to determine β arrestin2 albumen in conjunction with antibody or the part of β arrestin2 albumen.Preferable, the transcriptional level of described detection β arrestin2 or the reagent of expression are selected from: the antibody of the anti-β arrestin2 of specificity; Or the primer of specific amplification β arrestin2 mRNA.

As optimal way of the present invention, described reagent is primer, but its specific amplification goes out β arrestin2mRNA or fragment.Preferred, described primer is that a primer is right, has the sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, and described primer amplification effect is very good.

As optimal way of the present invention, described reagent is the antibody of the anti-β arrestin2 of specificity, and described antibody can be monoclonal antibody or polyclonal antibody.Described antibody can be prepared by the known various technology of those skilled in that art.For example, the β arrestin2 albumen of purification can be applied to animal (as rabbit) to induce the generation of polyclonal antibody.Similarly, the cell of expression β arrestin2 albumen can be used to immune animal and produces antibody.Described antibody also can be monoclonal antibody.This type of monoclonal antibody can utilize conventional hybridoma technology to prepare.

Design at the specific probe of β arrestin2 gene is technology well known in the art, for example, prepare a kind of probe, it can be combined by specific site generation specificity on β arrestin2 gene, and be not combined with other gene specific beyond the β arrestin2 gene, and described probe has detectable signal.

Utilizing specificity also is technology well known in the art in conjunction with the method that the antibody of β arrestin2 albumen detects β arrestin2 protein expression situation in the analyte.The most frequently used for example is the Western engram technology.

Whether the present invention also provides the existence that is used for detecting β arrestin2 at analyte and the test kit of expression, and this test kit comprises: the reagent of specific recognition β arrestin2 albumen or its encoding gene or transcript.In addition, also can comprise in the described test kit for extracting required all ingredients such as DNA, PCR, hybridization, colour developing, include but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, colour developing liquid, washing liquid etc.In addition, also can comprise operation instructions and/or Nucleotide Sequence Analysis Software etc. in the described test kit.

I. material and method

Mice

β Arrestins disappearance and wild-type mice by professor R.J.Lefkowitz of Duke Univ USA be so kind as to give (DukeUniversity Medical Center, Durham, NC).β Arrestins crosses and expresses and wild-type mice backcrosses by this prepared in laboratory and with C57BL/6 that (preparation method is referring to Shi more than nine generations, Y., et al., " Critical regulation of CD4+T cell survival and autoimmunity by beta-arrestin 1; " Nature Immunol., 8,817-24 (2007)).The raising of animal and operation are all carried out according to the regulations of Shanghai school of life and health sciences laboratory animal committee of the Chinese Academy of Sciences.All animals are all raised under bioclean environment.Normal mouse is fed normal diet (Formulab 5008, and Labdiet 5053), and high fat mice is fed high lipid food (containing 55% fat constituent) (Harlan-Teklad 93075).Adenovirus (7 * 10 ⁹Virion/100 μ l normal saline) by tail vein injection db/db mice and C57BL/6 mice, it is special is expressed in liver.The mensuration of body weight and dietary amount is with reference to Netea, M.G., et al., " Deficiency of interleukin-18 in mice leads tohyperphagia, obesity and insulin resistance, " Nature Medicine, 12,650-6 (2006).

Immunoprecipitation and immunoblotting

After mice was put to death, tissue was taken out fast and is stored in the liquid nitrogen.Organize lysate to measure protein concentration by the homogenate acquisition and by the Bradford method.For immunoprecipitation, cell pyrolysis liquid adds corresponding antibody, after 4 ℃ of overnight incubation, adds proteinA or protein G gel beads, and 4 ℃ were continued to hatch 2 hours.Be combined in that albumen on protein A or the protein G gel beads separates by SDS-PAGE and the method by immunoblotting detects corresponding albumen after SDS sample-loading buffer eluting and 50 ℃ are handled 20 minutes.For immunoblotting, protein band excites far infrared fluorescence by IRDye800CW coupling two is anti-, and system obtains by the Odyssey far infrared image, and (NE carries out quantitative analysis on USA) for Li-Cor Bioscience, Lincoln at Scion Image software subsequently.Concrete grammar such as bibliographical information Gao, H., et al., " Identificationof beta-arrestin2 as a G protein-coupled receptor-stimulated regulator of NF-kappaBpathways; " Mol.Cell, 14,303-17 (2004); And Luan, B., et al., " Beta-arrestin2 functionsas a phosphorylation-regulated suppressor of UV-induced NF-kappaB activation, " EMBO J., 24,4237-46 (2005).

The plain tolerance of mouse islets and glucose tolerance experiment (GTTs and ITTs)

In glucose tolerance experiment, mice passes through lumbar injection after hungry 6 hours method afford glucose stimulates, get blood at corresponding time point by the tail point and measure blood glucose with Roche Accu-chek blood glucose meter, with rat/mouse insulin ELISA kit measurement insulin level.In the insulin resistant experiment, mice directly through the method afford insulin stimulating of lumbar injection, is got blood at corresponding time point by the tail point and measures blood glucose with Roche Accu-chek blood glucose meter.

Data statistics

All data all are expressed as meansigma methods ± average standard variance.Multi-group data relatively adopts the method for ANOVA, and carries out the average check by Bonferroni.Relatively employing Student ' s t check between two groups of independent datas.The P value is less than 0.05 expression significant difference.

Cell transfecting and plasmid

Human liver cell Hep3B (available from ATCC) LipofectAMINE (Invitrogen) transfection.In the transfection, CMV-β-Gal is used for mending flat DNA amount.Total length Akt and Src are cloned into the pcDNA3 carrier of 5HA or Flag modification.AktTyr315/326Ala is cloned on the pcDNA3 carrier of 5Flag modification equally.The structure of β arrestin2 and mutant thereof is with reference to Gao, H., et al., " Identification of beta-arrestin2 as a G protein-coupledreceptor-stimulated regulator of NF-kappaB pathways; " Mol.Cell, 14,303-17 (2004).DNA sequence is by its correctness of sequence verification.

Antibody

β Arrestins rabbit source multi-resistance (A1CT and A2CT) by professor R.J.Lefkowitz of Duke Univ USA be so kind as to give (DukeUniversity Medical Center, Durham).Akt, Akt (phosphorylation Thr308), Akt (phosphorylation Ser473), GSK3-/β (phosphorylation Ser21/9), Foxo1, Foxo1 (phosphorylation Ser256), Src, phosphorylation Tyr and IR antibody are available from Cell Signaling Technology, Inc. (Danvers, MA, U.S.A.).GSK3-/β antibody is available from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.) .PP2 and Wortmannin available from Sigma-Aldrich (St.Louis, MO, U.S.A.).

Detection kit

Rat/mouse insulin ELISA test kit is available from Linco Research, Inc. (St.Charles, MO, U.SA.) .PI3-kinases ELISA kit is available from Echelon Biosciences, Inc. (Salt Lake City, Utah, U.S.A.) rat/mouse glucagon ELISA test kit, free fatty acid, triglyceride, with the cholesterin detection reagent box available from WAKO Chemicals USA (Richmond, VA). mice epinephrine ELISA test kit is available from USCNLife Science (China, Wuhan).Akt kinases test kit is available from Cell Signaling Technology, Inc..

The structure of adenovirus and injection

Target gene fragment (Gal, β-arrestin2, β-arrestin2 1-185 and β-arrestin2 186-409) clone is assembled in the multiple clone site of PShuttle-CMV carrier for expression of eukaryon, recombinate by prokaryotic cell recombination system and PAdEasy-1, and in HEK293 cell (available from ATCC), produce virus (Stratagene after linearization, La Jolla, CA, U.S.A.).The virus of CsCl density gradient purification is with 7 * 10 ⁹The amount of virion/100 μ l normal saline is passed through tail vein injection.

Real-time quantitative RT-PCR

Real time quantitative PCR method such as bibliographical information Kang, J., et al., " A nuclear function of beta-arrestin1in GPCR signaling:regulation of histone acetylation and gene transcription; " Cell, 123,833-47 (2005).HPRT mRNA level is as confidential reference items.The primer: mice β arrestin1-forward, 5-AAGGGACACGAGTGTTCAAGA (SEQ ID NO:1)-3, oppositely, 5-CCC GCT TTC CCA GGTAGA C-3 (SEQ ID NO:2); Mice β arrestin2-forward, 5-GGC AAG CGC GAC TTT GTA G-3 (SEQ ID NO:3), oppositely, 5-GTG AGG GTC ACG AAC ACT TTC-3 (SEQ ID NO:4); Mice HPRT-forward, 5-CCT GCT GGA TTA CAT TAA AGC ACT G-3 (SEQ ID NO:5), oppositely, 5-TTC AAC ACT TCG AGA GGT CCT-3 (SEQ ID NO:6).

Tissue specificity glucose metabolism and insulin behavior

The glucose production of primary hepatocyte is to measure according to the method for document description, Yang, Q., et al., " Serumretinol binding protein 4 contributes io insulin resistance in obesity and type 2 diabetes, " Nature, 436,356-62 (2005), glucose (GO) Assay Kit (available from Sigma) is used to detect the level of glucose.It is to measure according to the method for document description that Ex vivo glucose is taken in, Dubois, M.J., et al., " TheSHP-1protein tyrosine phosphatase negatively modulates glucose homeostasis, " Nature Med.12,549-56 (2006), and, 2-[ ³H]-deoxy-D-glucose was from PerkinElmer (Waltham, MA, U.S.A.).

Hepatocellular former be commissioned to train foster

Liver is after collagenase fully digests, the separation of primary hepatocyte and cultivation are carried out (Arkan according to the method for document record, M.C., et al., " IKK-beta links inflammation to obesity-induced insulin resistance; " Nature Med.11,191-8 (2005)).

H/E dyeing

Mouse tissue takes out and spends the night by 4% formalin fixed, and paraffin embedding, section and H/E dyeing are all carried out according to Standard Operating Procedure, and observe photograph by optical microscope.

II. embodiment

Embodiment 1. β arrestin2 are obviously downward modulation in type-II diabetes model mice sample

In the process of the novel targets of searching for curative interference insulin resistant, the inventor is seeking those tissues the insulin resistant mice, the different molecule of expression in the tissue that especially plays an important role in insulin replies.By these research, the inventor finds that the expression of β arrestin2 in the liver of type-II diabetes model db/db mice and muscle (liver and muscle are that insulin is regulated and the important tissue of functionating) is reduced.

Shown in Figure 1A, in the diabetic mice db/db mice that genotype causes, the protein level of β arrestin2 be expressed in liver, tangible downward modulation is arranged in the muscle, protein level descends 50% at liver, descends 20% at muscle.Corresponding mRNA level descends 50% at liver, at muscle decline 20% (Figure 1B).Because liver and muscle are main insulin induction tissues, observed these results suggest β arrestin2 is at insulin resistant or insulin resistant relevant disease, as working in the type-II diabetes.

Participate in insulin resistant in order further to evaluate β arrestin2, in the insulin resistant mouse model that high fat diet is induced, repeat same experiment.The insulin resistant mouse model that high fat diet is induced is by providing high fat diet (HFD) (containing 55% fat) to produce, concrete grammar is with reference to Yang, Q.et al., " Serum Retinol Binding Protein 4Contributes To Insulin Resistance In Obesity And Type 2Diabetes; " Nature 436,356-62 (2005).In these mices, the protein expression level of β arrestin2 also has tangible downward modulation at liver in the muscle, and protein level has descended 60% at liver, at muscle 30% (Fig. 1 C) that descended.Corresponding mRNA level has descended 50% at liver, at muscle 30% (Fig. 1 D) that descended.On the other hand, no matter be in db/db mice or the HFD inducing mouse, there is not significant change as the albumen of β arrestin2 in brain or the lung and the expression of mRNA level in its fat and the hetero-organization thereof.In addition, corresponding downward modulation (Figure 1A is also arranged in the liver that is expressed in these insulin resistant mices of β arrestin1 and the muscle, 1B), but modulation is much smaller than β arrestin2 under it, and hint β arrestin1 may not too work in insulin related disorders.

Embodiment 2. β arrestin2 are obviously downward modulation in the type-II diabetes patient samples

Except mouse model, the inventor also detects the protein level of β arrestin2 in 8 groups of type-II diabetes patient samples, diabetic compares with normal person's sample according to sex and age pairing, and being expressed in of β arrestin2 has tangible downward modulation (Fig. 8 A) in the patient samples.

Therefore, the inventor finds that β arrestin2 has obvious downward modulation in type-II diabetes patient samples and model mice sample, this has hinted that β arrestin2 plays a part in insulin signaling pathway certain, and its disappearance is closely related with the generation of insulin resistant and type-II diabetes.Implement really so following description.

Embodiment 3. β arrestin2 disappearance causes insulin resistant

In order to assess β arrestin2 possibility role in insulin signaling transduction and type-II diabetes, the inventor has investigated β-arrestin2 in the insulin behavior of whole body and the function in the glucose metabolism.The β arrestin2 gene delection mice (β arr2-ko) that makes up lacks β arrestin2 expresses (Fig. 9 A, on) but grows normally, and compares with brood wild-type mice and to have identical body weight and dietary amount (Fig. 9 A).β arr2-ko mouse blood sugar (Fig. 2 B, 2E) similar to the brood mice of wild type under starvation with insulin level (Fig. 2 A), but arr2-ko mouse blood sugar (Fig. 2 B) and insulin level (Fig. 2 A) be apparently higher than wild-type mice under the situation of feeding again, and hint β arr2-ko mice is for the higher demand of insulin and the defective of glucose dynamic equilibrium.

In the type-II diabetes patient samples, also found similar result.Fig. 8 B shows that β arrestin2 expresses the obvious rising that the patient who significantly descends is accompanied by hungry blood glucose equally.

Because β arrestin2 knocks out the defective that causes glucose dynamic equilibrium, the inventor has studied the dynamic equilibrium whether expression that increases β arrestin2 can promote insulin function and glucose.β arrestin2 gene overexpression mice (β arr2-tg mice) is to produce by the β arrestin2 that the CMV promoter starts expressing human, by the expression of β arrestin2 in the immunoblotting detection β arr2-tg mouse liver, the result shows that its expression is twice (Fig. 9 B of wild type approximately; On).β arr2-tg mice develop is normal, and compares with brood wild-type mice and to have identical body weight and dietary amount (Fig. 9 B).β arr2-tg mouse blood sugar is identical with wild-type mice under starvation with insulin level, but under the situation of feeding again blood glucose and insulin level be starkly lower than wild-type mice (Fig. 2 C, 2D).These results show that β arr2-tg mice is regulated increasing of glucose dynamic equilibrium ability.

In order further to study the function of β arrestin2, the inventor has carried out insulin resistant and glucose tolerance test to β arr2-ko mice, β arr2-tg mice and the brood mice of their wild type.In glucose tolerance test, lumbar injection glucose (1 gram/kilogram) can cause wild-type mice blood glucose to raise rapidly and slowly returned to hungry level after 2 hours subsequently, has reflected the ability of wild-type mice insulin secretion and glucose clearance.And β arr2-ko mice show as blood sugar increasing higher (Fig. 2 E, 2G), in glucose tolerance test, the reply glucose load insulin secretion in β arr2-ko mice, also hint that than higher (Fig. 9 C) these mices are destroyed the sensitivity of insulin.This conjecture is further confirmed (FIGS by the insulin resistant experiment.2F，2G)。

In the insulin resistant test, lumbar injection insulin (1U/kg) can cause wild-type mice and β arr2-ko mouse blood sugar to descend rapidly and slow recovery subsequently (Fig. 2 F).But it is more not obvious that β arr2-ko mice shows as the blood glucose reduction, and the duration of effect of the hypoglycemia of insulin is short, and (Fig. 2 F 2G), has reflected the reduction of the plain sensitivity of β arr2-ko mouse islets.

On the contrary, glucose sugar tolerance GTTs (1gkg ^-1) and insulin resistant experiment ITTs (1Ukg ^-1) confirmed that β arr2-tg mice shows as the raising of insulin sensitivity (Fig. 2 H, I, J and Fig. 9 C).

In a word, these body inner analysis show, lack β arrestin2 and damage insulin sensitivity, and increase the protein level raising insulin sensitivity of β arrestin2.These results have proved the importance of the insulin sensitivity of β arrestin2 and glucose dynamic equilibrium.

Liver, muscle and fat are main insulin sensitivity tissues, and the generation of they and insulin resistant and type-II diabetes is closely related.From top conclusion as can be seen, being expressed in the fat of β arrestin2 changes little (Figure 1A-1D).In addition, β arr2-ko mice, β arr2-tg mice and wild-type mice be in white adipose tissue weight, the adipose cell size, blood plasma free fatty acid, triglyceride and cholesterol aspect all do not have difference (Fig. 9 H-J).These results show that the influence of the fatty tissue of β arrestin2 is far smaller than it to the influence of liver and muscle.

It is the marked feature (Arkan that insulin resistant and glucose do not tolerate that insulin produces the forfeiture and the sugared minimizing of taking in of muscle that suppress to glycogen, M.C.et al., " IKK-beta links inflammation to obesity-induced insulinresistance; " Nat.Med., 11,191-8 (2005) .).Therefore, the inventor is to β arr2-ko mice, and β arr2-tg mice and wild-type mice liver insulin sensitivity detect.PEPCK and G6P are two key enzymes of liver glyconeogenesis.Under the starvation, their expression in β arr2-ko mouse liver apparently higher than wild-type mice, but in β arr2-tg mouse liver, significantly descend (Fig. 9 K).Former β arr2-ko mouse liver cell background glycogen of being commissioned to train foster is than the wild-type mice height, and insulin is to the inhibitory action not obvious (Fig. 9 L) of glycogen generation.β arr2-tg mice primary hepatocyte then has opposite phenomenon (Fig. 9 L).These results show that β arrestin2 can promote the insulin sensitivity of liver.

In addition, the inventor uses 2-deoxidation-D- ³The H-glucose is studied the absorption of the background glucose in the muscle.The sugar of background is taken in β arr2-ko mice, do not have difference in β arr2-tg mice and the wild-type mice musculus soleus, but the effect of insulin there are differences.But the glucose of insulin stimulating is taken in and be starkly lower than (2 times) wild-type mice in β arr2-ko mice, and β arr2-tg mice is then apparently higher than the brood mice of (1.5 times) wild type (Fig. 9 M).In EDL muscle, also found identical phenomenon (data not shown).These results show that β arrestin2 can promote the insulin sensitivity of muscle.

Embodiment 4. expresses β arrestin2 can recover insulin sensitivity

In order to determine whether β arrestin2 can prevent and improve the insulin resistant that obesity causes, after β arr2-ko mice, β arr2-tg mice and the high fat diet of the brood mice of their wild type through 8 weeks, β arr2-ko mice weight increase 50%, their wild type is brood mice weight increase 30%, and β arr2-tg mice weight increase 10% (Fig. 9 D) only, and in the nursing process, these dietary amount basic identical (Fig. 9 E).Further insulin resistant (1.5Ukg ^-1) and glucose tolerance (1.5gkg ^-1) test has proved that also β-arrestin2 can alleviate the insulin resistant (Fig. 3 A-F) that produced by high fat feeding, thereby alleviation is fat and the development of the fat insulin resistant that causes.

Above result shows that β arrestin2 transgenic can become a kind of effectively preventing and therapeutic agent.But, be with it as prevention and therapeutic agent, β arrestin2 need be imported in the cell, for example just can make its effective functionating by direct injection β arrestin2 albumen or with the expression vector transfered cell.Liposome or similar techniques can be used to mediate the importing of β arrestin2, and these technology are quite ripe.

In order in cell, to express β arrestin2, the polymerized nucleoside acid sequence of coding β arrestin2 can be built on the expression vector (as plasmid vector or viral vector such as retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus, CMV, SV40, or RSV), and use suitable initiating sequence, for example initiating sequence (beta-actin promoter) or tissue-specific initiating sequence (as liver, muscle, fatty specific initiating sequence) widely.If use viral vector, virus can infection cells and at cell inner expression β arrestin2.If the use plasmid vector then can adopt liposome or similar techniques to make plasmid enter cell and expresses β arrestin2.Liposome or similar techniques are quite ripe.

For example, whether insulin resistant and type-II diabetes are had prevention or therapeutic effect in order to study assessment β arrestin2, in the db/db diabetic mice, inventor's employing detects by the method that tail vein injection contains the adenovirus of β arrestin2.This recombinant viral vector causes the expression of β arrestin2 in its liver to increase by 3 times after importing to the db/db diabetic mice, and the body weight of mice and dietary amount do not change (Fig. 9 F and G).The blood glucose of β-arrestin2 adenovirus injection mice descends (Fig. 3 G) to some extent than contrast mice (for example receiving the mice of the adenovirus injection of expressing beta galactosidase (galactosidase) gene (β Gal)) under the feeding situation.In addition, insulin resistant and glucose tolerance test have proved that also the injection of β arrestin2 adenovirus can improve glucose tolerance and insulin resistant (Fig. 3 H-J).

Above result shows, improve β arrestin2 expression and not only can promote the insulin sensitivity of normal mouse and the insulin resistant that prevention of obesity is induced, and can recover the insulin sensitivity of type-II diabetes mice, show that β arrestin2 can be used as the New Policy of prevention and treatment insulin resistant and type-II diabetes.

Embodiment 5. β arrestin2 interact to promote the Akt activity by mediation Akt/Src

For the treatment potentiality of all-round exploration β arrestin2, the inventor has investigated the molecular mechanism in β arrestin2 activity.At molecular level, insulin resistant mainly is because the defective of peripheral tissues's insulin signaling pathway causes, in order to set forth the mechanism that β arrestin2 influences insulin resistant at molecular level, the inventor studies the insulin signaling pathway of β arr2-ko mice, β arr2-tg mice and the brood mouse liver of their wild type and muscle.

Fig. 4 A has shown in wild-type mice, β arr2-ko mice and β arr2-tg mice, under the insulin stimulating in the liver PI3K activity all improved about 3 times, insulin shows similar in all mouse genotypes to the activation of PI3K, hint β arrestin2 does not participate in the activation of PI3K directly.Inequality is that the activity of Akt but has significant difference in the liver in wild-type mice, β arr2-ko mice and β arr2-tg Mus therewith.The vitro kinase experiment is presented in wild-type mice and the β arr2-ko mouse liver difference of insulin-induced Akt activity (for example, GSK3 phosphoric acid being turned to pGSK3, Figure 10 A).Further, shown in Fig. 4 B and 10B, Akt characterizes its activity in the phosphorylation of 308 serines or 473 threonine under insulin stimulating, insulin stimulating causes the raising of Akt phosphorylation about 3 times in the liver in contrast, and this raising of the phosphorylation of Akt has reduced 50% in β arr2-ko mouse liver, and the phosphorylation of Akt has improved 2 times (Fig. 4 C and 10B) in β arr2-tg mouse liver on the contrary.The inventor has also observed similar phenomena (Figure 10 F, and G) in the muscular tissue of β arr2-ko mice and β arr2-tg Mus.Accordingly, compare with wild-type mice, GSK β and Foxo1 are significantly descended in the liver of β arr2-ko mice by the level of Akt phosphorylation, then obviously improve in β arr2-tg mice on the contrary (Fig. 4 B, and C).

In addition, external source imports β arrestin2 albumen and has also found similar effect.After the db/db mice is by tail vein injection β arrestin2 adenovirus, behind the insulin stimulating in the liver phosphorylation of Akt increase by 2 times, the phosphorylation of GSK3 β and Foxo1 also increases (Fig. 4 D, and 10C)).Therefore, the adenovirus expression carrier that comprises β arrestin2 coded sequence can effectively increase β-arrestin2 in intracellular expression, and the β arrestin2 that expresses has function.So this class carrier just can be used as effective prevention and treatment reagent.

More than these data show β arrestin2 can promote phosphorylation and the activation of the Akt of insulin stimulating, but do not influence the activity of PI3K.The function of these phenomenon promptings β arrestin2 mediation is the downstream at PI3K.

The activity of Akt is closely regulated and control by PI3K.Existing bibliographical information, the Akt of Src can improve Akt serine/threonine phosphorylation greatly at 315 and 326 s' tyrosine phosphorylation, and is the prerequisite of Akt activation.Corresponding, under the situation that Src inhibitor PP2 exists, Akt tyrosine and serine/threonine phosphorylation are by significant suppress (Fig. 4 E) in the Hep3B hepatocyte.In addition, when the Src phosphorylation site on the Akt suddenlyd change (Tyr315/326Ala) thus after making its forfeiture tyrosine phosphorylation, the activity of the Akt of insulin stimulating reduces (Figure 10 E).On the other hand, the inhibitor Wortmannin of PI3K handles the Hep3B cell can effectively suppress Akt serine/threonine phosphorylation and tyrosine phosphorylation, and hint Akt and Src are in the downstream of PI3K (Fig. 4 E).

How not rely on PI3K and exercise its function in order to understand β arrestin2, the inventor has detected the tyrosine phosphorylation of Akt in wild-type mice, β arr2-ko mice and the β arr2-tg mouse liver.Insulin stimulating causes raising (Fig. 4 F of about 2 times Akt tyrosine phosphorylation in contrast, last figure, swimming lane 2 is than swimming lane 1, with Figure 10 D), but Akt tyrosine phosphorylation 40% (swimming lane 4 is than swimming lane 2) that descended improved 1.5 times of (Fig. 4 F, figure below on the contrary in β arr2-tg mice in β arr2-ko mouse liver, swimming lane 4 is than swimming lane 2 and Figure 10 D).These results have hinted that β arrestin2 can promote Akt tyrosine phosphorylation and final Akt serine/threonine phosphorylation and the activity of promoting of Src mediation.

The classical function of bibliographical information β arrestin2 is to promote different MAPKs as joint albumen, as the activation of ERK1/2 and JNK3.The inventor infers that similar mechanism may also be present in the insulin signaling transduction of β arrestin2 mediation.For detecting this probability, at first the method with immunoprecipitation has detected Akt, β arrestin2, Src three's mutually combining in the C57BL/6 mouse liver.Fig. 5 A shows, when endogenic β arrestin2 is got off by immunoprecipitation, Akt, Src are also simultaneously precipitated to get off, when endogenic Akt is got off by immunoprecipitation, corresponding β arrestin2, Src is the while precipitated get off (Fig. 5 A, lane 4) also, and Akt therefore can reach a conclusion, β arrestin2, Src form a complex (Fig. 5 A) in vivo.

In order to verify the joint protein function of β arrestin2 in Akt/ β arrestin2/Src complex, inventor endogenic Akt of immunoprecipitation in β arr2-ko mice, β arr2-tg mice and the brood mouse liver of wild type detects the Src under the co-precipitation.Express Akt and the Src (Fig. 5 B, and C) of similar level in these mouse livers.What is interesting is that the Src in β arr2-ko mouse liver under the co-precipitation obviously is less than wild type (Fig. 5 B, swimming lane 2 is than swimming lane 1 and Figure 11 A).Opposite in β arr2-tg mouse liver the Src under the co-precipitation obviously more than wild type (Fig. 5 C and Figure 11 B).Therefore, Akt, β arrestin2 is necessary for mediation Akt and Src interaction in vivo.

In order to determine β arrestin2 and Akt and the interactional zone of Src, made up the mutant that a series of β arrestin2 block, carried out studying (Fig. 5 D) with the site that the method for co-immunoprecipitation mutually combines to the three.The experiment of co-immunoprecipitation shows, knock out the forfeiture that β arrestin2N end (amino acid/11-185) and C end (amino acid/11 85-409) can both cause β arrestin2 to be combined with Akt, do not influence β arrestin2-Akt interaction (Figure 11 C) and knock out N terminal amino acid 1-119.Therefore, the 120-185 of β-arrestin2 at least, one of 185-409 and 120-409 amino acids zone are that Akt is in conjunction with necessary.

And for Src, knock out the forfeiture that β arrestin2N end (amino acid/11-185) directly causes β arrestin2 to be combined with Src.On the contrary, β arrestin2 comprises the same and Src existence interaction of peptide Duan Zexiang wild type of amino acid/11-185, and these results show that Src combines with the N end of β arrestin2, and binding site is positioned at the zone of β arrestin21-185 amino acids.

Above result show β arrestin2 stride the zone of 1-185 may be only in conjunction with Src not in conjunction with Akt (Fig. 5 E).Therefore, the truncate mutation body that contains the 1-185 amino acids (β arrestin21-185) of β arrestin2 is used to check in the insulin signaling transduction, and it is essential that the Akt that β arrestin2 relies on for Src activates.When β arrestin21-185 crossed expression in the Hep3B cell, β arrestin21-185 was by having suppressed Akt/Src and be combined (Fig. 5 F, swimming lane 2 is than swimming lane 1) with endogenic β arrestin2 competition.Simultaneously, the Akt phosphorylation that insulin stimulating causes and the GSK3 β in Akt downstream and FoxO1 phosphorylation are subjected to obvious suppression (Fig. 5 G, swimming lane 4 is than swimming lane 2).Therefore prompting, β arrestin2 1-185 has destroyed Akt/ β arrestin2/Src signal complex as dominant negative mutant, thus the Akt that has suppressed insulin stimulating activates.These digital proofs the importance that forms as the protein mediated Akt/ β of joint arrestin2/Src complex of β arrestin2.

Embodiment 6. β Arrestin2 are connected to Akt/Src the IR of insulin-activation

Top result of study prompting insulin stimulating may be played a role in the formation of Akt/ β arrestin2/Src complex, in order to verify this probability, the inventor has also detected formation the replying insulin stimulating of Akt/ β arrestin2/Src complex.Fig. 6 A and Figure 12 B show that the lumbar injection insulin can cause in the C57BL/6 mouse liver Akt/Src to mutually combine and Akt/ β arrestin2 mutually combines, these show that in conjunction with time to time change insulin stimulating promotes the formation of Akt/ β arrestin2/Src albumen composition really.

The formation of Akt/ β arrestin2/Src albumen composition is subjected to the stimulation of insulin to hint this complex or its constituent directly and Insulin receptor INSR (IR) combination.Really, the interaction of β arrestin2 and IR is also confirmed (Figure 12 A) by in vitro tests.The immunoprecipitation of IR has disclosed β arrestin2 and has been enrolled into (Fig. 6 β and Figure 12 C) on this receptor in time dependent mode.The processing of the interaction partners Wortmannin of β arrestin2 and IR insensitive (Figure 12 D), thus show that this is in conjunction with not relying on and PI3K.

Insulin receptor INSR is combined with IR with β arrestin2 with the variation that Akt and Src mutually combine and is changed similar (Fig. 6 B and Figure 12 C).Insulin receptor INSR and Akt and Src mutually combine and are subjected to PI3K mediation (Figure 12 D) under the normal condition.Discover that further the combination of Insulin receptor INSR and Akt and Src also depends on the expression of β arrestin2 (Fig. 6 C), these phenomenons show that β arrestin2 is that Akt/Src is combined necessary with Insulin receptor INSR.What is more important, this is combined β arrestin2186-409 with IR but is not combined the mutant of (Fig. 6 D) with Akt/Src, exercise its function as dominant negative mutant, the Akt that the overexpression in the Hep3B hepatocyte suppresses insulin stimulating activates (Fig. 6 E).In a word, these results show that β arrestin2 can mediate the formation that has active IR/Akt/ β arrestin2/Src signal transduction complex under the insulin stimulating.

The expression of embodiment 7. β arrestin2 1-185 or 186-409 mutant causes insulin resistant

Studies show that β arrestin2 1-185 and the β arrestin2 186-409 of front can both suppress the Akt activation of insulin stimulating in dominant negative mode.Find based on this whether the inventor has further studied these two mutants can cause the interior insulin resistant of body.The method of C57BL/6 mouse tail vein injection adenovirus is come the specific β Gal that expresses in liver, β arrestin2 1-185 and β arrestin2 186-409, the blood glucose that contains β arrestin2 1-185 and β arrestin2 186-409 adenovirus injection mice in the time point of all mensuration with respect to β Gal adenovirus injection mice obviously raise (Fig. 7 A).Further glucose (1g/kg) tolerance and insulin (1U/kg) Resistance Test have proved that directly β arrestin2 1-185 and the injection of β arrestin2186-409 adenovirus have also damaged the tolerance (Fig. 7 B-D) of mice to glucose and insulin.Db/db diabetic mice tail vein injection contains β Gal, and the adenovirus of β arrestin2 1-185 and β arrestin2 186-409 has also obtained similar result (Figure 13).These results show that the β arrestin2 sudden change physical ability that can destroy IR/Akt/ β arrestin2/Src signal complex worsens insulin sensitivity and causes insulin resistant, thereby promote the development of diabetes.

In sum, above experimental result shows, be subjected to insulin stimulating after, β arrestin2 is as the interaction of scaffolding protein mediation Akt/Src, and next Akt/Src complex and Insulin receptor INSR (IR) is connected to form a signal complex.β arrestin2 is an essential scaffolding protein in this signal complex, because the knocking out or destruction that afunction can cause the transduction of this signal complex, insulin signaling and final insulin metabolism activity of β arrestin2.Therefore, the formation of the IR/Akt/ β arrestin2/Src complex of insulin stimulating is most important for insulin signaling transduction and insulin sensitivity.This complex disappearance or defective may cause insulin resistant and type-II diabetes.

Well-known β arrestin2 has abundant expression in liver and muscle, but up to the present whether β arrestin2 participates in insulin signaling transduction, and β arrestin2 at insulin resistant and type-II diabetes developing function takes place and also is not elaborated.The inventor is interior and external experiment by a series of bodies, found for the first time the existence of the signal complex that the IR/Akt/ β arrestin2/Src of a β arrestin2 mediation is new, and disclosed this complex insulin signaling pathway and in insulin resistant and type-II diabetes develop irreplaceable important function.In this signal complex, the effect that β arrestin2 has played scaffolding protein has mediated Akt, the interaction of Src and Insulin receptor INSR.The downward modulation of the expression of β arrestin2 or sudden change have caused the depolymerization of this signal complex, the reduction of the Akt activity that Src relies on, and finally promoted the generation of insulin resistant and the formation of type-II diabetes.Therefore, these phenomenons show that the pathological characteristics of β arrestin2 and insulin resistant and type-II diabetes exists direct physiology to contact, and provide a new theoretical direction and research target spot for the control of insulin resistant and type-II diabetes.

Insulin signaling transduction causes some scaffolding proteins as, IRS, GAP, and Grb2 and Shc etc. is enrolled into Insulin receptor INSR.The activation of the tyrosine kinase activity of Insulin receptor INSR receptor autophosphorylation causes series reaction, as the autophosphorylation of receptor, and the phosphorylation of scaffolding protein such as IRS, the activation of the recruitment of PI3K and activation and Akt.

Insulin signaling transduction path with respect to previously described classics, as if the Akt of the β arrestin2 mediation that the inventor finds activate different, it does not influence the activity of PI3K by Src activation Akt, thereby has proposed a brand-new insulin signaling transduction path.But the two also may not be certain to separate fully.For example, β arrestin2 may not influence the activity of PI3K, and it may promote the film migration of Akt, and the film migration can promote that Akt activates.β arrestin2 may be that the Akt that the Src of insulin stimulating relies on activates necessary.The phosphorylation in Akt tyrosine site may help the migration of Akt film and the serine/threonine phosphorylation by upstream kinases PDKs initiation afterwards.IRS, PI3K and PDKs also may be present in the signal complex of newfound this IR/Akt/ β arrestin2/Src, are conducive to signal transduction (Fig. 7 E) thereby this two signal paths is integrated into a signal network.

Studies show that in the former foster fibroblast of being commissioned to train before,. β arrestin1 rather than β arrestin2 can be in conjunction with the IGF-1 receptors, promote its endocytosis and strengthen map kinase moral phosphorylation and the synthetic (Dalle of DNA that the IGF-1 receptor relies on, S., et al., " Insulin and insulin-like growth factor I receptors utilize different G protein signalingcomponents, " J.Biol.Chem.276,15688-95 (2001); Lin, F.T., et al., " beta-arrestinsregulate mitogenic signaling and clathrin-mediated endocytosis of the insulin-like growthfactor I receptor; " J.Biol.Chem., 273,31640-3 (1998)).Yet the glucose metabolism of insulin-mediated and the activity of map kinase but do not rely on β arrestin1.The present invention finds it is the glucose metabolism activity that β arrestin2 rather than β arrestin1 can participate in insulin signaling transduction and downstream.The difference of β arrestin2 and β arrestin1 function may be because they cause with the different of Akt binding ability.

No matter still be that external β arrestin2 can both be combined with Akt in vivo, β arrestin1 is combined with Akt and is then wanted weak a lot (Fig. 5 A) with respect to β arrestin2.Therefore, β arrestin1 and β arrestin2 may regulate and control IGF and insulin signal path separately, have embodied both and have coordinated and not redundant function.

The formation that β arrestin2 has mediated IR/Akt/ β arrestin2/Src signal complex is the mechanism of its regulation and control insulin signaling pathway.For the signal complex, its universal feature is that the disappearance of any one component in the complex can produce similar effects.At this signal complex of IR/Akt/ β arrestin2/Src, IR, the influence of the insulin signaling transduction of Akt and Src, insulin resistant and type-II diabetes is more clearly, this research has given elaboration to the function of β arrestin2 again, and β arrestin2 plays an important role in metabolic disease by the characteristic that influences the signal complex.For example, the disappearance that sudden change or disappearance β arrestin will cause complex to form, and finally cause severe insulin resistance and type-II diabetes.Consistent is that the data that obtain from diabetic mice and type-II diabetes patient samples show the down-regulated expression of β arrestin2 rather than Akt or Src (data not shown) therewith.Prior, give the symptom that diabetic mice β arrestin2 can recover its insulin sensitivity and alleviate type-II diabetes, show that β arrestin2 can be used as the reagent of prevention and treatment type-II diabetes.Generally speaking, β arrestin2 has played the effect of hinge in the signal complex, and can be used as the reagent of a kind of diagnostic flag or a kind of prevention or treatment insulin resistant or type-II diabetes.

For example, a kind of diagnostic kit of the β of comprising arrestin2 antibody can be used to detect the protein expression level of β arrestin2 in the slicer.And these tissues might not be confined to liver, muscle and fat.Immunoblotting, immunoprecipitation, SABC, several different methods such as immunofluorescence can be employed.Perhaps, a kind of diagnostic kit that can comprise the reverse transcriptional PCR primer of energy specific amplified β arrestin2mRNA can be used to detect the mRNA expression of β arrestin2 in the slicer.And these tissues might not be confined to liver, muscle and fat.The mRNA expression can be quantitative with different detection methods, and as real-time RT-PCR, method such as densitometry analysis can be used to the result is carried out quantitatively behind the electrophoresis.Compare to diagnose patient's insulin resistant level with β arrestin2 albumen in the normal specimens or mRNA expression, relatively low β arrestin2 expression indicates that this patient may have insulin resistant, thereby becomes the candidate of β arrestin2 or other treatment.

Classical IR signal path is mainly recruited IR scaffolding protein such as IRS, GAP, Grb2, and Shc, the formation of their mediation a series of signal complex.On the other hand, the signal complex of β arrestin mediation is considered to only limit to GPCRs.Yet the inventor finds that β arrestin2 also can participate in the IR signal path by the formation of mediation IR/Akt/ β arrestin2/Src signal complex.And the formation of this albumen composition is subjected to insulin stimulating regulation and control, hints that this complex works in the insulin activity.The signal complex of β arrestin2 mediation is the new inherent component of a kind of brand-new discovery, Insulin receptor INSR signal path, and a novel targets that can be used as diagnosis, prevention or treatment insulin resistant and relevant disease such as type-II diabetes is provided.

In sum, the IR/Akt/ β arrestin2/Src signal complex of β arrestin2 mediation is in the insulin signaling transmission and keep in the whole body insulin sensitivity and play an important role, and its disappearance or defective will cause insulin resistant.Therefore, β arrestin2 is the target spot of a kind of very potential diagnosis, prevention and treatment insulin resistant and relevant disease thereof.

Find that based on these part of the present invention is embodied in utilizes β-arrestin2 to carry out the diagnosis of insulin resistant.According to embodiments of the invention, detect the expression of β-arrestin2 in the testing sample (for example slicer of liver or muscle).Detection can utilize the antibody (monoclonal or polyclone) of β arrestin2, testing result with compare from the result of normal specimens.Perhaps utilize arrestin2 mRNA level in the quantitative testing sample of reverse transcriptional PCR.These the reaction in for detection of antibody and the reverse transcriptional PCR primer below embodiment and experimental technique in described.

If the expression of β-arrestin2 is lower than normal subjects, then the object of surveying may have insulin resistant or tend to produce insulin resistant.These objects will become the prevention of the insulin resistant of intervening based on β-arrestin2 and the patient for the treatment of.

Embodiments more of the present invention relate to reagent and the method for prevention and treatment insulin resistant and relevant disease thereof.According to embodiment of the present invention, these reagent can comprise the expression vector that contains coding β arrestin2 sequence, and carrier can be plasmid vector or viral vector, viral vector can be retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.The initiating sequence of expression vector can be the CMV initiating sequence, SV40 initiating sequence, RSV initiating sequence, β-actin initiating sequence or tissue specificity initiating sequence.The tissue specificity initiating sequence can be that fatty tissue is special, and muscle specific or liver are special.

If the use plasmid vector then can adopt liposome or similar techniques to make plasmid enter cell and expresses β arrestin2.If use viral vector, virus can infection cells and at cell inner expression β arrestin2, thereby do not need transfection reagent.

These reagent can be used for preventing or treat the relevant disease of insulin resistant.These reagent can be given the patient via any suitable approach with an effective dosage, comprise oral, external, injection, etc.These reagent can be used for treating mammal, and are especially human.An effective dosage is the amount that can reach the effect of wanting.The effective dosage of any decision is general technology and patient's body weight, the age, and health, administration number of times waits relevant.

This invention superiority is embodied in the following aspects: the IR/Akt/ β arrestin2/Src signal path of the brand-new β arrestin2 mediation of finding has represented other approach that a kind of insulin activates Akt.β arrestin2 has played crucial effects when this insulin signaling pathway of regulation and control.Therefore, the expression of detection β arrestin2 or activity provide diagnostic method easily for insulin resistant.Similar, the reagent that β arrestin2 expresses in cell plasmid-lipidosome complex or the β arrestin2 adenovirus expression carrier of β arrestin2 coded sequence (as comprise) can be used for increasing and prevention or treatment insulin resistant and relevant disease thereof will be hopeful to be used to.Using the prevention of method of the present invention and reagent or treat insulin resistant can be very effective in prevent diabetes or similar disease, because these reagent directly act on the signal path of β arrestin2 mediation, can be treated before the symptom development.

Because description of the invention is based on the embodiment of limited quantity, after the content of having read above-mentioned instruction of the present invention, those skilled in the art can make various changes or modification to the present invention, and these equivalent form of values fall into the application's appended claims institute restricted portion equally.

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＜110〉Shanghai Inst. of Life Science, CAS

＜120〉be used for diagnosis, method and the reagent of prevention and treatment insulin resistant

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Claims (7)

1. a β arrestin2 albumen or the purposes adjusted on it are for the preparation of the compositions of prevention or treatment mammal insulin resistant or insulin resistant relevant disease;

Described insulin resistant or insulin resistant relevant disease are selected from: type-II diabetes, hypertension, disorders of lipid metabolism, cardiovascular disease, obesity, Hyperinsulinism, metabolic arthritis disease, or glucose intolerance.

2. purposes as claimed in claim 1 is characterized in that, the upward adjustment of described β arrestin2 albumen comprises the expression vector of the polynucleotide of coding β arrestin2.

3. purposes according to claim 2 is characterized in that, described expression vector is plasmid vector.

4. purposes according to claim 3 is characterized in that, described expression vector is viral vector.

5. purposes according to claim 4 is characterized in that, described viral vector is selected from down group: retrovirus, adenovirus, herpesvirus, vaccinia virus or adeno-associated virus.

6. purposes according to claim 1 is characterized in that, described mammal is the people.

7. purposes as claimed in claim 1 is characterized in that, described compositions is used for promoting that Insulin receptor INSR/Akt/ β arrestin2/Src signal transduction complex forms.

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