CN103585629B - A kind of target preventing and treating insulin resistant and relevant disease thereof - Google Patents
A kind of target preventing and treating insulin resistant and relevant disease thereof Download PDFInfo
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- CN103585629B CN103585629B CN201210289347.3A CN201210289347A CN103585629B CN 103585629 B CN103585629 B CN 103585629B CN 201210289347 A CN201210289347 A CN 201210289347A CN 103585629 B CN103585629 B CN 103585629B
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Abstract
The present invention relates to a kind of new target drone preventing and treating insulin resistant and relevant disease thereof。Demonstrate hprl receptor first and body insulin sensitivity is had raising effect, lower PRLR and can substantially reduce insulin sensitivity。Therefore, PRLR itself or on it adjustment can be used in increase insulin sensitivity；And based on the above-mentioned functions of PRLR, the material increasing insulin sensitivity can be screened。
Description
Technical field
The invention belongs to biotechnology and pharmaceutical art；More particularly it relates to an the new target drone of preventing and treating insulin resistant and relevant disease thereof。
Background technology
Insulin resistant refers to that the insulin in blood circulation is for fat, liver, the decline of glycolipid metabolism ability of regulation and control in muscle。Insulin resistant (insulin insensitivity) has become one of threat that public health is the most serious。It causes the imbalance of body glucose dynamic equilibrium and has close contacting with glucose intolerance, obesity, disorders of lipid metabolism, hypertension and cardiovascular disease。After insulin resistant occurs, body is compensate the decline of insulin sensitivity and the substantial amounts of insulin of improper generation, directly results in hyperinsulinism symptom in blood circulation。The final result of insulin resistant development is the generation of diabetes。
At present, whole world diabetics presents increasing trend, and wherein 90% patient is type 2 diabetes mellitus。Type 2 diabetes mellitus be mainly characterized by occur insulin resistant, be embodied in muscle and fatty tissue glucose absorption reduce, in liver glycogenolysis increase。Insulin resistant is the one of the main reasons that non-alcoholic fatty liver disease occurs, and is likely to further result in more serious hepatic disease generation, even hepatocarcinoma。
Insulin resistant Producing reason is the decline that insulin stimulating is responded by Insulin receptor INSR。Under usual state, insulin in blood circulation is fatty, liver, the Insulin receptor INSR identification on muscle cell surface, Insulin receptor INSR be activated after phosphorylated insulin receptor substrate protein (IRSproteins), the activation of the further mediate downstream PI3K signal path of phosphorylation of IRS albumen。PI3K signal path has mediated the metabolic function that insulin is main。
Insulin signaling pathway is a complexity the signal network by high precision regulation and control。In this path, PI3K energy phosphorylation PI4,5-P2 produce film anchoring molecule PI3,4,5-P3 of Akt and PDKs。Akt, will by PDKs phosphorylation and activate once move on film。The kinases in the Akt phosphorylation downstream successively of activation and transcription factor, thus it is movable to mediate most insulin metabolism。In insulin signaling pathway, the defect of the key protein such as such as Akt all will cause serious insulin resistant。The forfeiture of Akt gene knockout or kinase activity all directly results in insulin resistant。
The hormone of body different tissues secretion, for instance leptin, adiponectin, in regulating insulin sensitivity process, play very important effect。Because hormone plays a role at the receptor of cell surface mainly by conjunction with it, so hormone receptor is the key factor determining the reaction of final specific physiologic。
Prolactin antagonist is the hormone mainly secreted by antepituitary, but, its receptor hprl receptor is to the adjustment effect of insulin sensitivity unclear。
Summary of the invention
It is an object of the invention to provide a kind of new target drone preventing and treating insulin resistant and relevant disease thereof。
In a first aspect of the present invention, it is provided that a kind of hprl receptor or the purposes adjusted on it, for preparing the compositions increasing insulin sensitivity。
In a preference, described hprl receptor is: (a) GenBank accession number: the polypeptide of aminoacid sequence shown in NP000940.1；Or (b) through by the aminoacid sequence of (a) polypeptide and replacement, lacks or add one or several (such as 1-20, preferably 1-10, more preferably 1-5) aminoacid and there is the polypeptide derivative by (a) of (a) polypeptide function；Or the amino acid sequence homology of (c) and (a) polypeptide (is preferably higher than 90% higher than 80%, more preferably higher than 95%, more preferably higher than 98%) and there is the polypeptide derivative by (a) of (a) polypeptide function。
In another preference, described compositions is used for preventing, alleviate or treat insulin resistant or insulin resistant relevant disease。
In another preference, described insulin resistant or insulin resistant relevant disease include, but is not limited to: diabetes (are preferably type 2 diabetes mellitus), hypertension, disorders of lipid metabolism, cardiovascular disease, fat, Hyperinsulinism, metabolic arthritis disease, or glucose intolerance。
In another preference, described compositions is additionally operable to:
Increase Insulin receptor INSR (IR) or AKT phosphorylation level；Or
Increase glucose-tolerant and Scavenging activity。
In another preference, the upper adjustment of described hprl receptor is selected from (but not limited to): the expression vector of the polynucleotide comprising coding hprl receptor and the expression regulation sequence being operatively connected with it is (preferably, for pCMV-HA, this expression vector can be used for converting cell and expressing hprl receptor)；Or the compositions (such as food, beverage, health product, medicine) that leucine lacks。
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 lower group: retrovirus, adenovirus, herpesvirus or vaccinia virus。
In another aspect of this invention, it is provided that the purposes of hprl receptor, for increasing the target of the medicine of insulin sensitivity as screening。
In another aspect of this invention, it is provided that a kind of screening method for increasing the potential material of insulin sensitivity, described method includes:
(1) candidate substances is contacted with the system comprising (as expressed) hprl receptor；
(2) the detection candidate substances impact on hprl receptor；
If described candidate substances can improve expression or the activity of hprl receptor, then show that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
In a preference, step (1) including: in test group, candidate substances is joined in the system comprising (as expressed) hprl receptor；And/or
Step (2) including: detects expression or the activity of hprl receptor in the system of test group, and compares with matched group, and wherein said matched group is the system comprising (as expressed) hprl receptor without described candidate substances；
If in test group the expression of hprl receptor, activity statistically higher than (be preferably significantly higher than, as high by more than 20%, it is advantageous to high by more than 50%；More preferably high by more than 80%) matched group, indicate that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
In another preference, described system, in step (1), also comprises (as expressed) Insulin receptor INSR (IR) or AKT；And,
In step (2), also include: detection Insulin receptor INSR (IR) or AKT phosphorylation level, if Insulin receptor INSR or AKT phosphorylation level increase (preferably dramatically increase, as increased by more than 20%, it is advantageous to increase by more than 50%；Increase by more than 80% more preferably), then show that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
In another preference, described system is selected from: cell system (or cell culture system) (such as the cell of recombinant expressed Insulin receptor INSR, HepG2 cell or primary hepatocyte), subcellular fraction system, solution system, organizational framework, organ systems or animal system。
In another preference, described candidate substances includes, but is not limited to: for the over-express vector (plasmid) of hprl receptor design, receptor stimulating agent, micromolecular compound。
In another preference, described method also includes: the potential material obtained is carried out further cell experiment and/or animal experiment, to select further from candidate substances and to determine the compositions useful for increasing insulin sensitivity。
In another aspect of this invention, it is provided that a kind of method increasing mammalian islet element sensitivity, described method includes: raise expression or the activity of hprl receptor in mammal body。
The other side of the present invention, due to this disclosure, is apparent to those skilled in the art。
Accompanying drawing explanation
Fig. 1, prolactin antagonist regulate insulin sensitivity in vitro。
Cell infection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR) 48h (A), HepG2 cell hprl receptor interference fragment (+PRLRRNAi) or comparison fragment (-PRLRRNAi) process 48h, primary hepatocyte infects gland virus expression for bobby pin (sh) the structure RNA (+Ad-shPRLR) of hprl receptor or comparison virus (-Ad-shPRLR) 72h (B), then adds (+ins) or is not added with (-ins) 100nM insulin (Ins) and stimulates 20min。Data are at least through three independent experiments, meansigma methods ± standard error。Data significance calculates (p < 0.05) by t-test。(A and B) p-IR, p-AKT, PRLR albumen。Wherein, upper figure is immunoblotting, and figure below is that each band of upper figure is quantitatively schemed relative to actin。
Fig. 2, in vivo utilize adenovirus Ad-PRLR process LAN hprl receptor improve insulin sensitivity。
C57BL/6J male mice tail vein injection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR), detect hprl receptor in liver when 12 days and express (A, upper figure is immunoblotting, figure below is that hprl receptor is quantitatively schemed relative to actin), blood glucose and insulin level in serum (B and C is detected when 14 days, B is blood sugar level, C is insulin level), calculate HOMA index (D), glucose tolerance test and insulin resistant experiment (E) is done when the 8th day or the 10th day, insulin signaling pathway (F in liver is detected when 12 days, left figure is immunoblotting, right figure is that each band of left figure is quantitatively schemed relative to actin)。Animal experimental data, at least through 2 independent experiments, often organizes n=5-6, meansigma methods ± standard error。Data significance calculates (p < 0.05) by t-test。
Fig. 3, in vivo utilize adenovirus Ad-shPRLR reduce hprl receptor reduce insulin sensitivity。
C57BL/6J male mice tail vein injection adenovirus process LAN for hprl receptor bobby pin (sh) structure RNA (+Ad-shPRLR) or comparison virus (-Ad-shPRLR), detect hprl receptor in liver when 7 days and express (A, upper figure is immunoblotting, figure below is that hprl receptor is quantitatively schemed relative to actin), blood glucose and insulin level in serum (B and C is detected when 14 days, B is blood sugar level, C is insulin level), calculate HOMA index (D), glucose tolerance test and insulin resistant experiment (E) is done when the 3rd day or the 4th day, insulin signaling pathway (F in liver is detected when 12 days, left figure is immunoblotting, right figure is that each band of left figure is quantitatively schemed relative to actin)。Animal experimental data, at least through 2 independent experiments, often organizes n=5-6, meansigma methods ± standard error。Data significance calculates (p < 0.05) by t-test。
Fig. 4, hprl receptor increase insulin sensitivity by activating ERK。
HepG2 cell infection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR) 48h, C57BL/6J male mice tail vein injection adenovirus process LAN hprl receptor (+Ad-PRLR) or 12 days (A of green fluorescent protein (-Ad-PRLR), hprl receptor and p-erk express, upper figure is immunoblotting, figure below is that upper figure band is quantitatively schemed relative to actin), C57BL/6J male mice tail vein injection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR), then before within the 7th day, being ITT (B), 5h injects PD98059 (+PD) or comparison (-PD), within 9th day, beat insulin detection hepatic insulin signal path change (C, left figure is immunoblotting, right figure is that each band of left figure is quantitatively schemed relative to actin), A diagram data is at least through three independent experiments, meansigma methods ± standard error, A-C animal experimental data is at least through 2 independent experiments, often organize n=5-6, meansigma methods ± standard error, data significance calculates (p < 0.05) by t-test or one-wayANOVA。
Fig. 5, hprl receptor process LAN can improve the sensitivity under vivo and vitro insulin-resistant states。
HepG2 cell infection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR), glucamine (+GlcN) or be not added with after (-GlcN) stimulate 18h after 30h, 100nM insulin stimulating 20min (A and B, left figure is immunoblotting, right figure is that each band of left figure is quantitatively schemed relative to actin), hprl receptor is protein expression analysis (C in wild type (wt) and db/db (db), upper figure, immunoblotting, figure below is that each band of upper figure is quantitatively schemed relative to actin), C57BL/6Jdb/db male mice tail vein injection adenovirus process LAN hprl receptor (+Ad-PRLR) or green fluorescent protein (-Ad-PRLR), blood glucose and insulin level in serum (D and E is detected when 11 days and 7 days, D is blood sugar level, E is insulin level), glucose tolerance test and insulin resistant experiment (F) is done when the 7th day or the 9th day, A and B diagram data is at least through three independent experiments, meansigma methods ± standard error, C-F animal experimental data is at least through 2 independent experiments, often organize n=5-6, meansigma methods ± standard error, data significance calculates (p < 0.05) by t-test or one-wayANOVA。
Fig. 6, reduction hprl receptor are expressed and can be lacked the insulin sensitivity increase caused by external reduction leucine in vivo。
C57BL/6J male mice gives normally (+Leu) or leucine shortage grain (-Leu) and feeds seven days, HepG2 cell gives normally to train base (+Leu), or leucine lacks training base (-Leu) and processes 12h (A and B, A, hprl receptor mRNA, B, hprl receptor albumen), HepG2 cell normally trains base (+Leu) giving, or leucine shortage training base (-Leu) transfects hprl receptor interference fragment (+PRLRRNAi) or (-PRLRRNAi) 36h before processing 12h, then 100nM insulin stimulating 20min (C), C57BL/6J male mice gives normally (+Leu) or leucine and lacks grain (-Leu) nursing, then tail vein injection adenovirus process LAN for hprl receptor bobby pin (sh) structure RNA (+Ad-shPRLR) or comparison virus (-Ad-shPRLR), within 3rd day, it is glucose tolerance test and insulin resistant experiment (D and E, D, GTT, E, ITT) insulin detection hepatic insulin signal path change (F) within the 5th day, is beaten, A-C figure cell experiment data are at least through three independent experiments, meansigma methods ± standard error, A, B, D, F figure animal experimental data is at least through 2 independent experiments, often organize n=5-6, meansigma methods ± standard error, data significance calculates (p < 0.05) by t-test or one-wayANOVA。
The hprl receptor increase that Fig. 7, leucine cause when lacking is by GCN2/S6K approach。
GCN2+/+Or GCN2-/-Male mice gives normally (+Leu) or leucine shortage grain (-Leu) and feeds seven days (A and B, A, hprl receptor mRNA, B, hprl receptor albumen), C57BL/6J male mice tail vein injection adenovirus process LAN HA labelling sustained activation S6K1 (+Ad-CA-S6K1) or green fluorescent protein (-Ad-CA-S6K1) are given afterwards and are lacked 7 days (C and D of diet with leucine, C, hprl receptor mRNA, D, hprl receptor albumen), GCN2-/-Male mice leucine lacks diet 7 days, every day lumbar injection rapamycin (+Rapa) or (-Rapa) 1mg/Kg (E, hprl receptor, p-S6, p-S6K1 protein expression, upper figure is immunoblotting, and figure below is quantitatively scheme), regulatory mechanism illustraton of model (F)。Animal experimental data, at least through 2 independent experiments, often organizes n=5-6, meansigma methods ± standard error, and data significance calculates (p < 0.05) by t-test。
Detailed description of the invention
The present inventor, through extensive and deep research, demonstrates hprl receptor (prolactinreceptor, PRLR) first and body insulin sensitivity has raising effect, lowers PRLR and can substantially reduce insulin sensitivity。Therefore, PRLR itself or on it adjustment can be used in increase insulin sensitivity；Further, based on the above-mentioned functions of PRLR, the material increasing insulin sensitivity can be screened。
PRLR and application thereof KTHYJHFK
Hprl receptor (PRLR) is membrane receptor one species specific, high-affinity, and research finds that it belongs to 1 type cytokines receptor family, lacks endogenous kinase domain, relies primarily on intracellular kinase and carry out conducted signal。Have now been found that three kinds of hprl receptors, be divided into short, medium-sized and elongated according to their length。Elongated hprl receptor is expressed extensively, and the present inventor is also mainly derived from elongated for the research of hprl receptor signal path。Hprl receptor is mainly through regulating protein kinase (ERK) and JAK/STAT5 signal path functionating outside active cell。
Hprl receptor has expression in mammiferous most tissues and cell, participates in regulating a lot of physiological process, works including well-known to those skilled in the art in breeding。But, whether hprl receptor participates in the important mechanisms of this affecting glucose steady-state adjustment of insulin sensitivity, it is not clear that。
The inventors discovered that PRLR increases the New function of insulin sensitivity。The present inventor finds under study for action, and the expression at mouse liver process LAN or suppression hprl receptor can strengthen or reduce the insulin sensitivity of mice；In vitro experiment adopt human liver cell HepG2 or Mus primary hepatocyte to observed same phenomenon。Then the inventors discovered that, it has been found that ExtracellularRegulatedProteinKinases (ERK) has played important function in this course。When insulin resistant model db/db mice and insulin sensitivity enhancing (as leucine lack mice) in, the expression of hprl receptor reduces respectively or raises。Strengthen or weaken the insulin sensitivity when expression of hprl receptor can recover both accordingly。Finally the inventors discovered that, leucine lacks the expression being regulated hprl receptor by generalcontrolnonderepressible (GCN) 2/mammaliantargetofrapamycin (mTOR)/S6K1 signal path。These researchs illustrate hprl receptor and regulate the New function of insulin sensitivity, and hprl receptor can as the novel targets for the treatment of insulin resistant medicine。These results specify that liver hprl receptor New function in regulating insulin sensitivity and illustrate the expression of trophic factor adjustment hprl receptor。These results also imply that the hprl receptor probability of potential target as treatment insulin resistant and relevant disease non-alcoholic fatty liver disease thereof。
Therefore, based on the new discovery of the present inventor, the invention provides the purposes of PRLR albumen, for preparing the compositions increasing insulin sensitivity；Or for screening the material increasing insulin sensitivity。
Increasing for prevention, alleviation or treatment insulin insulin resistance or insulin resistant relevant disease of insulin sensitivity is useful, these diseases include, but is not limited to: type 2 diabetes mellitus, hypertension, disorders of lipid metabolism, cardiovascular disease, fat, Hyperinsulinism, metabolic arthritis disease, or glucose intolerance。
In the present invention, PRLR albumen used can be naturally-occurring, and such as it can be separated or be purified from mammal。Additionally, described PRLR albumen can also be artificial preparation, such as restructuring PRLR albumen can be produced according to conventional genetic engineering recombinant technique。Preferably, the present invention can adopt the PRLR albumen of restructuring。
Any applicable PRLR albumen is used equally to the present invention。Described PRLR albumen includes PRLR albumen or its bioactive fragment of total length。Preferably, the aminoacid sequence of described PRLR albumen can be substantially the same with the sequence shown in GenBank accession number NP000940.1。
The aminoacid sequence of the PRLR albumen formed through the replacement of one or more amino acid residues, disappearance or interpolation is also included within the present invention。PRLR albumen or its bioactive fragment include the alternative sequence of a part of conserved amino acid, and the described sequence replaced through aminoacid has no effect on its activity or remains the activity of its part。Suitably replacing aminoacid is technology well known in the art, and described technology can be implemented and guarantee not change the biological activity of gained molecule easily。These technology make those skilled in the art recognize, in general, change single amino acids in the unwanted regions of a peptide species essentially without changing biological activity。See Watson etc., MolecularBiologyofTheGene, the four editions, 1987, TheBenjamin/CummingsPub.Co.P224。
The bioactive fragment of any PRLR albumen can be applied in the present invention。Here, the bioactive fragment of PRLR albumen is meant that referring to as is a peptide species, and it still can keep all or part of function of PRLR albumen of total length。Under normal circumstances, described bioactive fragment at least keeps the activity of the total length PRLR albumen of 50%。Under still more preferential conditions, described active fragment can keep the activity of the 60% of total length PRLR albumen, 70%, 80%, 90%, 95%, 99% or 100%。
The present invention may be used without modified or improvement PRLR albumen, such as, can adopt the PRLR albumen modified to promote the effect of its half-life, effectiveness, metabolism and/or albumen or improve。The described PRLR albumen through modifying or improve can be the conjugate of a kind of PRLR albumen, or it can comprise that be replaced or artificial aminoacid。The described PRLR albumen through modifying or improve can be have less common ground with naturally occurring PRLR albumen, but also can increase insulin sensitivity, and will not bring other harmful effect or toxicity。It is to say, any bioactive version not affecting PRLR albumen can be used in the present invention。
Aminoacid sequence according to PRLR albumen can draw its corresponding nucleotide coding sequence easily。Preferably, the nucleotide sequence of described PRLR albumen can be substantially the same with the sequence shown in GenBank accession number NP000940.1。
Upper adjustment of PRLR and application thereof
Based on the above-mentioned new discovery of the present inventor, the invention provides the purposes of the upper adjustment of a kind of PRLR, be used for preparing increase insulin sensitivity。
As used herein, the upper adjustment of described PRLR includes accelerator, agonist etc.。The material of the transcription and translation of the activity of any PRLR of raising albumen, the stability of maintenance PRLR albumen, the expression of promotion PRLR albumen, the secretion of promotion PRLR albumen, prolongation PRLR albumen effective acting time or promotion PRLR is used equally to the present invention, as can be used for increasing the active substance of insulin sensitivity。
Optimal way as the present invention, the upper adjustment of described PRLR includes, but is not limited to: the expression vector of the polynucleotide comprising coding hprl receptor and the expression regulation sequence being operatively connected with it, this expression vector can convert cell and express hprl receptor；Or the food that leucine lacks。
As the optimal way of the present invention, the upper adjustment of described PRLR albumen includes, but is not limited to: can express expression vector or the expression constructs of (preferred process LAN) PRLR after proceeding to cell。Generally, described expression vector comprises a box gene, and described box gene contains the gene encoding PRLR and the expression regulation sequence being operatively connected with it。Described " being operatively connected " or " being operably coupled to " refers to such a situation, and namely some part of linear DNA molecule can regulate or control the activity of same linear DNA molecule other parts。Such as, if promoter controls transcribing of sequence, then it is operably coupled to coded sequence exactly。
In the present invention, PRLR polynucleotide sequence can be plugged in recombinant expression carrier。As long as can replicate in host and stable, any plasmid and carrier may be used to the present invention。One key character of expression vector is to usually contain origin of replication, promoter, marker gene and translation to control element。
Method well-known to those having ordinary skill in the art can be used for building the DNA sequence containing PRLR and the suitable expression vector transcribing/translate control signal。These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.。Described DNA sequence can be effectively connected in the suitable promoter in expression vector, to instruct mRNA to synthesize。Conversion carrier also includes ribosome binding site and the transcription terminator of translation initiation。
Compositions
Present invention also offers a kind of compositions, it contains effective dose (such as 0.000001-50wt%；Preferably 0.00001-20wt%；More preferably, 0.0001-10wt%) described PRLR albumen or its on adjust, and pharmaceutically acceptable carrier。
The compositions of the present invention can be directly used for increasing insulin sensitivity。Additionally, also can be used in combination with other therapeutic agent or adjuvant simultaneously。
Generally, can being formulated in by these materials in aqueous carrier medium nontoxic, inertia and pharmaceutically acceptable, wherein pH ordinarily be about 5-8, it is preferred that, pH is about 6-8。
As used herein, term " contains " and represents that various composition can be applied in mixture or the compositions of the present invention together。Therefore, term " mainly by ... composition " and " by ... form " be included in term " containing "。As used herein, term " effective dose " or " effective dose " refer to amount that is that people and/or animal can produce function or activity and that can be accepted by people and/or animal。
As used herein, the composition of " pharmaceutically acceptable " applies to people and/or mammal and without excessive bad side reaction (such as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio。Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent。
The compositions of the present invention contains the PRLR albumen of safe and effective amount and pharmaceutically acceptable carrier。This kind of carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof。Usual pharmaceutical preparation should match with administering mode, and the pharmaceutical composition of the present invention can be made into injection form, for instance is prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant。Described pharmaceutical composition should aseptically manufacture。The dosage of active component is therapeutically effective amount。The pharmaceutical preparation of the present invention may also be fabricated which slow releasing preparation。
PRLR albumen of the present invention or the effective dose adjusted on it can change with the order of severity etc. of the pattern of administration and disease to be treated。The selection of preferred effective dose can be determined (such as passing through clinical trial) by those of ordinary skill in the art according to various factors。Described factor includes but not limited to: described PRLR albumen or the pharmacokinetic parameter such as bioavailability, metabolism, half-life etc. adjusted on it；The order of severity of the disease that patient to treat, the body weight of patient, the immune state of patient, administration approach etc.。Generally, when the PRLR albumen of the present invention or on it adjust every day give with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals), gratifying effect can be obtained。Such as, by an urgent demand for the treatment of situation, several times dosage separately can be given every day, or dosage is reduced pari passu。
Present invention also offers a kind of method increasing insulin sensitivity, including the PRLR albumen or the adjustment on it that give experimenter's effective dose, it is preferred to use the PRLR albumen of restructuring。
The PRLR albumen of the present invention or the administering mode adjusted on it have no particular limits, it is possible to be whole body or local。Such as, the PRLR albumen of the present invention or adjust on it can pass through spinal cord intrathecal injection, lumbar injection, intravenous injection, is administered orally, the mode of subcutaneous injection, intradermal injection etc. give animal, it is preferable to spinal cord intrathecal injection。
After the purposes knowing described PRLR albumen, it is possible to adopt multiple method well known in the art that described PRLR albumen or its encoding gene or its pharmaceutical composition are delivered medicine to mammal。Preferably, the means of gene therapy can be adopted to carry out, such as directly PRLR albumen can be delivered medicine to experimenter by methods such as such as injections；Or, by certain approach, the ceneme (such as expression vector or virus etc.) carrying PRLR gene can be delivered on target spot, and so as to the PRLR albumen of expression activity。
As one embodiment of the present invention, can described PRLR albumen be directly administered in mammal (such as people), or, can the gene of coding PRLR albumen be cloned in suitable carrier (such as routine protokaryon or carrier for expression of eukaryon or viral vector such as herpesvirus vector or adenovirus vector) by conventional method, in described vector introduction to the cell that can express described PRLR albumen, described cell will be made to express PRLR albumen。Can by appropriate described cell being incorporated into the suitable position of body of mammals, it is achieved the expression of PRLR albumen。
The administering mode of the upper adjustment of PRLR albumen depends primarily on type and the characteristic of described upper adjustment, and this is that those skilled in the art can assess。
Screening increases the method for the potential material of insulin sensitivity
After knowing described PRLR albumen purposes in increasing insulin sensitivity, it is possible to screen the material of expression or the activity promoting PRLR based on this feature。
Therefore, the present invention provides the method for the potential material that a kind of screening can be used for increasing insulin sensitivity, and described method includes: candidate substances contacted with the system expressing PRLR；With the detection candidate substances impact on PRLR；If described candidate substances can improve expression or the activity of PRLR, then show that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
In the optimal way of the present invention, when screening, the change of expression or activity in order to be more easily observable PRLR, matched group also can be set, described matched group can be the system expressing PRLR without described candidate substances。
The described system expressing PRLR can be such as cell (or cell culture) system, and described cell can be the cell of endogenous expression PRLR；It can be maybe the cell of recombinant expressed PRLR。The described system expressing PRLR can also is that (but not limited to) subcellular fraction system, solution system, organizational framework, organ systems or animal system (such as animal model) etc.。
As the optimal way of the present invention, described method also includes: the potential material obtained is carried out further cell experiment and/or animal experiment, to select further and to determine the material actually useful for increasing insulin sensitivity。
The present invention has no particular limits for the detection method of the expression of PRLR albumen, activity or amount。Protein quantification or the half-quantitative detection technology of routine can be adopted, for instance (but not limited to): SDS-PAGE method, Western-Blot method, ELISA etc.。
On the other hand, present invention also offers the potential material that can be used for increasing insulin sensitivity adopting described screening technique to obtain。The material that these Preliminary screening go out may make up a screening storehouse, in order to people may finally therefrom filter out material that can be actually useful for increasing insulin sensitivity, thus for clinic。
Below in conjunction with specific embodiment, the present invention is expanded on further。Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention。The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition such as J. Pehanorm Brooker etc. are write, Molecular Cloning: A Laboratory guide, Science Press, the condition described in 2002, or according to manufacturer it is proposed that condition。Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight。
Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words。Additionally, any method similar or impartial to described content and material all can be applicable in the present invention。The use that preferably implementation described in literary composition and material only present a demonstration。
I. research method and technology path
Animal model is combined by the present invention with cell model, respectively with wild type C57BL/6J mice, HepG2 and primary hepatocyte for object of study, collect insulin resistant and prolactin receptor gene knocks out, the liver organization of process LAN model and cell sample, for corresponding research。Research method is based on qRT-PCR, WesternBlotting, molecular cloning etc.。
II. materials and methods
Laboratory animal and process
Male C 57 BL/6 J mouse is bought in Shanghai Si Laike Experimental Animal Center。GCN2 knocks out (knockout) (Gcn2-/-) and Leptinreceptor-deficient (db/db) mice respectively available from PennStateUniversity, USA and Nanjing of China university。The male mice of 8-10 week old carries out daily raising in Shanghai Si Laike Experimental Animal Center, and SPF condition is in strict conformity with following standard: single cage is cultivated, raising temperature 23 ± 3 DEG C, humidity 35 ± 5%。Within 12 hours round the clock, it is circulated。Before carrying out formally experiment, mice is raised by the common food meeting international standard being purchased from this animal center。
Matched group grain and leucine shortage group grain that formal experiment is used are purchased from ResearchDiets company of the U.S. (NewBrunswick, NJ), method for breeding is shown in description (XiaoF etc. before, LeucinedeprivationincreaseshepaticinsulinsensitivityviaG CN2/mTOR/S6K1andAMPKpathways.Diabetes60:746-756), in brief, mice is first with comprising whole amino acid whose matched group grain raising 7-10 days, after having adapted to this kind of matched group grain, these mices are randomly divided into 2 groups: matched group and leucine shortage group。Mice uses sacrificed by carbon dioxide mice after carrying out glucose-tolerant (GTT) or insulin resistant experiment (ITT)。
PD98059 is purchased from Promega company of the U.S., it is the inhibitor (DiPaolaR etc. of ERK, PD98059, aspecificMAPkinaseinhibitor, attenuatesmultipleorgandysfunctionsyndrome/failure (MODS) inducedbyzymosaninmice.PharmacolRes61:175-187), for injecting adenovirus process LAN hprl receptor or the mice of matched group process LAN green fluorescent protein。PD98059 process group mice, ITT test before 5 hours, tail vein injection 200ulPBS comprises 75uMPD98059, matched group injection 200ulPBS comprises 0.37%DMSO, PD98059 dosage is according to the report (NijharaR etc. of article before, Sustainedactivationofmitogen-activatedproteinkinasesanda ctivatorprotein1bythehepatitisBvirusXproteininmousehepat ocytesinvivo.JVirol75:10348-10358,2001)。In rapamycin treatment experiment, rapamycin is purchased from Chinese T autoBiothech company, first it is dissolved in dehydrated alcohol and is made into 10mg/ml liquid storage, before injection, it is diluted in 5%PEG400, in 5%Tween80 and 4% ethanol, final concentration of 0.4mg/ml, then lumbar injection 1mg/kg body weight, matched group injection solvent (GeY etc. as described before, mTORregulatesskeletalmuscleregenerationinvivothroughkina se-dependentandkinase-independentmechanisms.AmJPhysiolCe llPhysiol297:C1434-1444, 2009)。The animal protection of Chinese Academy of Sciences's nutrition science institute and the correlative detail using rules are all followed in the operation of all mouse experiments。
Primary cell separates, and cell is cultivated and processed
Primary cell is separated by potting compound protoenzyme, (WangQ etc. as described before, AbrogationofhepaticATP-citratelyaseprotectsagainstfattyl iverandameliorateshyperglycemiainleptinreceptor-deficien tmice.Hepatology49:1166-1175,2009)。The hepatocyte separated is resuspended in the DMEM of 10% (v/v) FBS, cultivates one day postoperative infection virus。
The HepG2 DMEM culture fluid containing 10% heat-inactivated fetal bovine serum, 100 mcg/ml streptomycins and 100 units per ml penicillins is cultivated。Condition of culture is 37 DEG C, 5%CO2。PD98059 process group, HepG2 first with PD98059 pretreatment 30min, then deposits at PD98059 and uses 100nm insulin stimulating 20min in case, and matched group is not added with PD process。
The cell model of insulin resistant utilizes HepG2 cell to cultivate 18 hours induced synthesis in the anteserum-less substrate containing 18mM glucamine (glucosamine), (SunC etc. as described above, SIRT1improvesinsulinsensitivityunderinsulin-resistantcon ditionsbyrepressingPTP1B.CellMetab6:307-319,2007)。Matched group training base (all aminoacid containing required) and leucine lack the compound method training base: add common DMEM in AminoAcid-freeDMEM (Invitrogen) and train whole aminoacid (wherein, leucine shortage training base is not added with leucine) contained in base。
HepG2 cell transfecting siRNA: the double-strand siRNA for people source hprl receptor orders from Chinese Shanghai Ji Ma company, cell utilizes Roche Holding Ag of Germany X-tremeGenesiRNA transfection reagent transfection 40pmol/LsiRNA, and matched group only adds transfection reagent。The sequence of siRNA is: forward: GAAGCAUUGUUCUAGACAATT (SEQIDNO:1)；Reverse: UUGUCUAGAACAAUGCUUCTT。
The generation of recombinant adenovirus and injection
Hprl receptor process LAN plasmid and CA-S6K1 recombinant adenovirus are the AdEasy utilizing Qbiogene companyTMVectorSystem produces。
The construction step of hprl receptor process LAN recombinant adenovirus is as follows: prolactin antagonist over-express vector pACT2 from Japan AkihikoYoshimura laboratory (referring to TakahoEndo etc., CIS1InteractswiththeY532oftheProlactinReceptorandSuppres sesProlactin-DependentSTAT5Activation.TheJournalofBioche mistry133:109-113,2002), with it, for template design primer, (sequence is: forward: CGGGGTACCATGTCATCTGCAC (SEQIDNO:2)；Reverse: CCGCTCGAGTCAGTGAAAGGA (SEQIDNO:3)) PCR obtains hprl receptor cDNA, it is connected in pshuttle-CMV (Qbiogene, Montreal, Canada) kpn-1 and xho-1 restriction enzyme site, after obtaining prlr-pshuttle plasmid, pme1 linearization for enzyme restriction and pAd-easy plasmid (Qbiogene, Montreal, Canada) restructuring, after recombinating successfully, by recombiant plasmid pac I enzyme action, transfection 293A cell (ATCC), treat cell rounding, wrap out virus, expand after wrapping out virus, then cscl density gradient ultracentrifugation, purification carries out follow-up test。
CA-S6K1 recombination adenovirus construction step is as follows: CA-S6K1 plasmid buys (numbering 8991) from Addgene, construction method is with hprl receptor process LAN virus, first it is cloned into kpn-1 and the xho-1 restriction enzyme site of pshuttle-CMV, then pme1 linearization for enzyme restriction and the restructuring of pAd-easy plasmid, after recombinating successfully, by recombiant plasmid pac I enzyme action, transfection 293A cell, treat cell rounding, wrap out virus, expanding after wrapping out virus, then cscl density gradient ultracentrifugation, purification carries out follow-up test。
Green fluorescent protein process LAN recombinant adenovirus is referring to YifuQiu etc., AcrucialroleforRACK1intheregulationofglucose-stimulatedI RE1 α activationinpancreatic β-cells.ScienceSignaling3 (106), ra7,2010。
Adenovirus process LAN scrambled (sequence is TTCTCCGAACGTGTCACGT (SEQIDNO:4), is not for the shRNA sequence of any albumen) or the shRNA sequence of specific against mouse hprl receptor are to utilize the BLOCK-iT of Invitrogen companyTMAdenoviralRNAiExpressionSystem produces, and concrete experimental procedure is with reference to the description of this product。Virus PBS dilutes, and 12 orifice plate consumptions are 107Pfu/ml, mouse tail vein injection is 109Pfu/ mice。
The construction method of shRNA carrier (hprl receptor knockdown adenovirus vector): the BLOCK-iT provided according to Invitrogen companyTMRNAiDesigner program, input elongated PRLRcds region sequence, the sequence that software output is suitable, composition sequence (Top:5'-CACCGCCACCTACCATAACTGATGTCGAAACATCAGTTATGGTAGGTGG C-3'(SEQIDNO:5), bottom:5'-AAAAGCCACCTACCATAACTGATGTTTCGACATCAGTTATGGTAGG TGGC-3'(SEQIDNO:6)), then according to BLOCK-iTTMAdenoviralRNAiExpressionSystemprotocol, is connected in pENTR by sequenceTMOn/U6 carrier (Invitrogen), then by the plasmid of successful connection and adenovirus vector pAd/BLOCK-iTM-DEST (Invitrogen) recombinates, and after recombinating successfully, by recombiant plasmid pac I enzyme action, transfects 293A cell, treats cell rounding, wrap out virus, expands after wrapping out virus, and then cscl density gradient ultracentrifugation, purification carries out follow-up test。
Blood glucose, the mensuration of serum insulin, glucose tolerance test (GTT) and insulin resistant experiment (ITT)
The mensuration of blood glucose and serum insulin is respectively with GlucometerElitemonitor and MercodiaUltrasensitiveRatInsulinELISAkit (ALPCODiagnostic) detection。
Glucose-tolerant test (GTT), C57BL/6 mice overnight starvation pneumoretroperitoneum injection 2g/kg glucose solution, blood glucose meter detection blood sugar level is then utilized respectively when 0,15,30,60,90,120min。
Insulin resistant test (ITT), the hungry 4 hours pneumoretroperitoneums of C57BL/6 mice inject 0.75U/kg insulin respectively, then utilize blood glucose meter detection blood sugar level respectively when 0,15,30,60,90,120min。
Being calculated as of insulin resistance index (HOMA-IR) [hungry glucose level (mmol/L)] × [hungry serum insulin (μ U/ml)]/22.5。
The detection of internal insulin signaling pathway
The hungry 6 hours insulin injections of mice, (WangQ etc. as described above, AbrogationofhepaticATP-citratelyaseprotectsagainstfattyl iverandameliorateshyperglycemiainleptinreceptor-deficien tmice.Hepatology49:1166-1175,2009)。After mouse anesthesia, postcava injection 2U/kg insulin, after 3min, liver organization takes a fritter, puts into-80 DEG C of Refrigerator stores standby after liquid nitrogen freezing。Tissue adds cell pyrolysis liquid, leach protein after collecting, then westernblot detects p-IR/t-IR, p-AKT/t-AKT expression。
Immunoblotting
Western blotting method (ChengY etc., Leucinedeprivationdecreasesfatmassbystimulationoflipolys isinwhiteadiposetissueandupregulationofuncouplingprotein 1 (UCP1) inbrownadiposetissue.Diabetes59:17-25) as described before。Primary antibodie (anti-p-IR, anti-IR, anti-p-AKT, anti-AKT, anti-p-ERK, anti-ERK (equal purchased from American CellSignalingTechnology company), anti-PRLR (purchased from SantaCruzBiotechnology)) 4 DEG C of overnight incubation, Special Proteins utilizes ECLPlus (AmershamBiosciences) to develop the color, and band brightness is by QuantityOne (Bio-RadLaboratories) statistics。
RNA extracts and relative RT-PCR
Hprl receptor mRNA level in-site is detected by RT-PCR, (XiaoF etc. as described above, LeucinedeprivationincreaseshepaticinsulinsensitivityviaG CN2/mTOR/S6K1andAMPKpathways.Diabetes60:746-756), sequence is:
Forward: 5 '-TGAGGACGAGCGGCTAATG-3 ' (SEQIDNO:7)；
Reverse 5 '-GGTGTGTGGGTTTAACACCTTGA-3 ' (SEQIDNO:8)。
Statistical analysis
Numerical value all represents with means standard deviation。Between two groups, whether difference has statistical significance and adopts double; two tail t between non-matching group to detect。Matched group, leucine shortage group and dietary restriction amount group, use Student-Newman-Keulstest after the difference one-wayANOVA detection between these three groups again。When p is < when 0.05, it is believed that have significant difference between group。
III. embodiment
Embodiment 1, hprl receptor regulate insulin sensitivity in vitro
In order to determine hprl receptor effect in regulating insulin sensitivity, the present inventor utilizes prolactin antagonist process LAN adenovirus vector to infect HepG2 cell and primary hepatocyte, detects the hprl receptor impact on the crucial constitutive protein Insulin receptor INSR (IR) of insulin signaling pathway two and AKT phosphorylation。Research before finds that multiple the acting on after rise hprl receptor is expressed of prolactin antagonist is enhanced, and the process LAN of hprl receptor can activate the intracellular signaling pathway that prolactin antagonist is relevant。
The inventors discovered that, in HepG2 cell and primary hepatocyte after process LAN hprl receptor, IR and the AKT phosphorylation level that insulin causes dramatically increases (Figure 1A), the effect in increasing insulin sensitivity of the prompting hprl receptor。Consistent with these discoveries, after being utilized respectively RNAi interference fragment and adenovirus process LAN shRNA reduction hprl receptor expression, IR and the AKT phosphorylation level (Figure 1B) that insulin stimulating causes can be reduced。
Embodiment 2, in vivo process LAN hprl receptor increase insulin sensitivity
In order to study hprl receptor adjustment effect to insulin sensitivity in vivo, the present inventor is to mouse tail vein injection hprl receptor process LAN adenovirus, first find in experimental mice liver, no matter rna level or protein level, the expression of Prlr has significant increase (Fig. 2 A) relative to matched group, then experimental mice is all remarkably decreased (Fig. 2 B) relative to matched group blood glucose at full abdomen and starvation, although insulin level in serum is more or less the same during full abdomen, but experimental mice insulin level in serum is remarkably decreased under starvation, consistent with this, hprl receptor process LAN mice group HOMA index is significantly reduced, in order to prove tolerance and the Scavenging activity of experimental mice glucose further, the present inventor has done glucose tolerance test and insulin resistant experiment, find that experimental mice injectable dextrose monohydrate is after 60 minutes, blood sugar level is substantially less than matched group (Fig. 2 E), after insulin injection, experimental mice blood glucose declines and is substantially less than matched group (Fig. 2 E)。
These results suggest that, hprl receptor process LAN can improve the insulin sensitivity of whole body。Consistent with this, the inventors discovered that, in hprl receptor process LAN group mouse liver, insulin (Ins) stimulates IR and the AKT phosphorylation level caused also to dramatically increase (Fig. 2 F)。Female Mus also obtains similar result。
Embodiment 3, in vivo reduction hprl receptor are expressed and are reduced insulin sensitivity
For the research hprl receptor effect to insulin sensitivity in vivo further, the present inventor utilizes hprl receptor knockdown adenovirus vector Tail Vein injection Mouse, it has been found that in liver, hprl receptor is mRNA level in-site or protein level is all remarkably decreased (Fig. 3 A) relative to matched group。Although the full abdomen blood glucose of mice and fasting glucose are all without notable change (Fig. 3 B), but insulin level in serum dramatically increases (Fig. 3 C) relative to matched group under two states, and HOMA index also increases (Fig. 3 D) to some extent。Consistent with these results, GTT and ITT is it is demonstrated experimentally that experimental mice glucose-tolerant and Scavenging activity all significantly reduce (Fig. 3 E), and IR and the AKT phosphorylation level that in liver, insulin stimulating causes is significantly reduced (Fig. 3 F)。
Therefore, reduce hprl receptor in vivo and express reduction insulin sensitivity。
Embodiment 4, hprl receptor process LAN adenovirus are in vivo by activating ERK increase insulin sensitivity
Studies have found that ERK is the downstream (PosnerBI etc. of hprl receptor signal path before；Prolactinreceptorsinratliver:possibleinductionbyprolacti n.Science188:57-59, 1975), and ERK signal path can regulate insulin response (GeY etc., mTORregulatesskeletalmuscleregenerationinvivothroughkina se-dependentandkinase-independentmechanisms.AmJPhysiolCe llPhysiol297:C1434-1444, 2009), these researchs imply that ERK regulates insulin sensitivity as the downstream of hprl receptor, so the present inventor have detected the hprl receptor impact on ERK phosphorylation。
The inventors discovered that, in hepG2 cell and liver after process LAN hprl receptor, ERK phosphorylation level dramatically increases (Fig. 4 A)。The effect in insulin sensitivity is regulated at hprl receptor in order to study ERK further, dosage (the NijharaR etc. that the present inventor reports according to article before, Sustainedactivationofmitogen-activatedproteinkinasesanda ctivatorprotein1bythehepatitisBvirusXproteininmousehepat ocytesinvivo.JVirol75:10348-10358, 2001) to the inhibitor PD98059 (PD) of mice internal injection ERK, detect the effect that the insulin sensitivity that hprl receptor process LAN increase causes by it increases。After ITT experiment finds hprl receptor process LAN, glucose clearance ability increases, and can significantly reduce this effect (Fig. 4 B) after PD98059 injection。Meanwhile, IR and the AKT phosphorylation increase that hprl receptor process LAN causes also is suppressed significantly (Fig. 4 C) after injection PD98059。Although only injection PD98059 group mouse islets element signal path is impaired, but the Scavenging activity not impact (Fig. 4 C) that PD98059 is on glucose。
Embodiment 5, hprl receptor process LAN can improve the sensitivity under vivo and vitro insulin-resistant states
Based on passing through the hprl receptor injecting process LAN or the discovery of the KnockDown hprl receptor adenovirus vector adjustment effect to insulin sensitivity, the present inventor speculates that hprl receptor is likely to the adjustment in participation insulin resistant or sensitivity increase process to sensitivity。
First, the present inventor reports (SunC etc. according to article before, SIRT1improvesinsulinsensitivityunderinsulin-resistantcon ditionsbyrepressingPTP1B.CellMetab6:307-319, 2007) HepG2 cell is processed with 18mM glucamine, cause insulin resistant cell model, then the present inventor detects the expression of hprl receptor in insulin resistant cell, find that glucamine process group hprl receptor is expressed and be remarkably decreased (Fig. 5 A), in glucamine process cell after process LAN hprl receptor, phosphorylation IR and AKT that significantly reverses glucose amine causes can decline (Fig. 5 B)。
Leptin receptor lacks (db/db) mice, is common insulin resistant mice model (ChengY etc.；Leucinedeprivationdecreasesfatmassbystimulationoflipolys isinwhiteadiposetissueandupregulationofuncouplingprotein 1 (UCP1) inbrownadiposetissue.Diabetes59:17-25), the present inventor have detected the expression of hprl receptor in db/db mice, consistent with in vitro results, relative to normal mouse, hprl receptor is expressed and is significantly reduced (Fig. 5 C)。
Whether it is the reason causing db/db mouse islets element signal to decline to study hprl receptor decline, the present inventor is viral to db/db mouse tail vein injection hprl receptor process LAN, although finding that the change of hprl receptor process LAN group mice insulin level in serum is little, but full abdomen and fasting glucose are all remarkably decreased (Fig. 5 D and Fig. 5 E), and glucose-tolerant and Scavenging activity also dramatically increase (Fig. 5 F)。
Embodiment 6, reduction hprl receptor are expressed and can be lacked the insulin sensitivity increase caused by external reduction leucine in vivo
Research before finds that, when leucine (leu) lacks, insulin sensitivity increases (KellyPA etc.；Theprolactin/growthhormonereceptorfamily.EndocrRev12:235-251,1991), so the present inventor detects the expression of hprl receptor in this insulin sensitivity increase situation, during relative to insulin resistant, hprl receptor expression declines (Fig. 5 A and Fig. 5 C), and hprl receptor mRNA and protein level are expressed in the HepG2 cell of leucine shortage training base process and the mouse liver of leucine shortage diet and dramatically increased (Fig. 6 A and Fig. 6 B)。
So the present inventor speculates, hprl receptor take part in adjustment when leucine lacks to sensitivity, in order to verify that this is assumed, the present inventor processes in leucine shortage and utilizes RNAi interference fragment to reduce hprl receptor expression in cell, it has been found that leucine can be stoped to lack phosphorylation IR and the AKT caused increases (Fig. 6 C)。For further checking, the present inventor lacks 7 days process mice (KellyPA etc.) middle injection knockdown hprl receptor adenovirus vectors at leucine diet, find similar with in vitro results, leucine lacks the glucose-tolerant caused and Scavenging activity increase significantly reduces (Fig. 6 D and Fig. 6 E) after knockdown hprl receptor, and after insulin stimulating, phosphorylation IR and AKT is significantly reduced (Fig. 6 F)。
The hprl receptor increase that embodiment 7, leucine cause when lacking is by GCN2/S6K approach
Although playing an important role in the insulin sensitivity that hprl receptor is when regulating leucine and lacking, but leucine shortage is to regulate the mechanism of hprl receptor expression and unclear。GCN2 is a kind of serineprotein kinase, function primarily as sensor (ZhangW etc., the MAPK/ERKsignalingregulatesinsulinsensitivitytocontrolglu cosemetabolisminDrosophila.PLoSGenet7:e1002429 of amino acid starvation；KodamaH etc.；Developmentofhyperglycaemiaandinsulinresistanceinconscio usgeneticallydiabetic (C57BL/KsJ-db/db) mice.Diabetologia37:739-744,1994；WekRC etc.；Juxtapositionofdomainshomologoustoproteinkinasesandhisti dyl-tRNAsynthetasesinGCN2proteinsuggestsamechanismforcou plingGCN4expressiontoaminoacidavailability.ProcNatlAcadS ciUSA86:4579-4583,1989), the present inventor speculates that GCN2 is likely to take part in adjustment when leucine lacks to hprl receptor。In order to verify this probability, the present inventor have detected the expression of hprl receptor after leucine lacks or normal diet is fed 7 days of GCN2 normal mouse and knock-out mice, consistent with expection, hprl receptor mRNA and protein expression that normal mouse causes when leucine lacks increase major part in GCN2 knock-out mice and are prevented from (Fig. 7 A and Fig. 7 B)。
Studying discovery, leucine lacks mTOR/S6K1 signal path in mouse liver and reduces (KellyPA etc.), and therefore the present inventor speculates that the mTOR/S6K1 activity of reduction is likely to take part in the expression of hprl receptor before。In order to verify this probability, the present inventor penetrates sustained activation S6K1 process LAN adenovirus in leucine shortage mice note, sees whether it can reduce the expression of prolactin antagonist when leucine lacks。Consistent with expection, in injection sustained activation S6K1 process LAN adenovirus mouse liver, hprl receptor mRNA and protein level relative comparison group significantly reduce (Fig. 7 C and Fig. 7 D)。Because find that leucine lacks the S6K1 activity reduction caused and depends on GCN2 before, imply that S6K1 is probably GCN2 and regulates the downstream that hprl receptor is expressed。This probability is by injection mTOR inhibitors rapamycin (WekSA etc. in the GCN2 knock-out mice of leucine shortage diet, Thehistidyl-tRNAsynthetase-relatedsequenceintheeIF-2alph aproteinkinaseGCN2interactswithtRNAandisrequiredforactiv ationinresponsetostarvationfordifferentaminoacids.MolCel lBiol15:4497-4506, 1995) verified further, find that GCN2 knock-out mice rapamycin injection group liver hprl receptor is expressed and be significantly higher than matched group (Fig. 7 E)。
Embodiment 7, drug screening
The first step: using HepG2 cell (or application primary hepatocyte) as being used for screening the cell model of the medicine increasing insulin sensitivity。
Second step: drug screening:
Test group: with the culture of the HepG2 cell (or application primary hepatocyte) that candidate substances processes；
Matched group: without the culture of the HepG2 cell (or application primary hepatocyte) that candidate substances processes。
Appropriate time after treatment, adopts the expression (WesternBlotting) of the hprl receptor albumen of conventional method mensuration HepG2 cell or the change of activity and corresponding insulin signaling pathway。If compared with matched group, expression or the activity of the hprl receptor albumen in test group significantly rise more than 30%, then illustrate that this candidate substances is the potential material increasing insulin sensitivity。
Using the adenovirus vector of process LAN hprl receptor albumen as candidate substances, join in HepG2 cell culture, observe the amount of hprl receptor albumen in born of the same parents, found that the amount of hprl receptor albumen dramatically increases more than 30%, thus indicate process LAN hprl receptor albumen adenovirus vector can as improve insulin sensitivity drug candidate。
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document。In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally。
Sequence table
Claims (9)
1. hprl receptor or on it adjust a purposes, for prepare increase insulin sensitivity compositions；The upper adjustment of described hprl receptor is the polynucleotide comprising coding hprl receptor and the expression vector of the expression regulation sequence being operatively connected with it。
2. purposes as claimed in claim 1, it is characterised in that described compositions is used for preventing, alleviate or treat insulin resistant or insulin resistant relevant disease。
3. purposes as claimed in claim 2, it is characterised in that described insulin resistant or insulin resistant relevant disease include: diabetes, hypertension, disorders of lipid metabolism, cardiovascular disease, fat, Hyperinsulinism, metabolic arthritis disease, or glucose intolerance。
4. purposes as claimed in claim 3, it is characterised in that described compositions is additionally operable to:
Increase Insulin receptor INSR or AKT phosphorylation level；Or
Increase glucose-tolerant and Scavenging activity。
5. purposes according to claim 1, it is characterised in that described expression vector is viral vector。
6. purposes according to claim 5, it is characterised in that described viral vector is selected from lower group: retrovirus, adenovirus, herpesvirus or vaccinia virus。
7. the method that a screening is used for increasing the potential material of insulin sensitivity, it is characterised in that described method includes:
(1) candidate substances is contacted with the system comprising hprl receptor；
(2) the detection candidate substances impact on hprl receptor；
If described candidate substances can improve expression or the activity of hprl receptor, then show that this candidate substances is to can be used for increasing the potential material of insulin sensitivity；
Described system is cell culture system。
8. method as claimed in claim 7, it is characterised in that step (1) including: in test group, candidate substances is joined in the system comprising hprl receptor；And/or
Step (2) including: detecting expression or the activity of hprl receptor in the system of test group, and compare with matched group, wherein said matched group is the system comprising hprl receptor without described candidate substances；
If the expression of hprl receptor, activity are statistically higher than matched group in test group, indicate that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
9. method as claimed in claim 7, it is characterised in that in step (1), also comprise (as expressed) Insulin receptor INSR (IR) or AKT in described system；And,
In step (2), also include: detection Insulin receptor INSR or AKT phosphorylation level, if Insulin receptor INSR or AKT phosphorylation level increase, then show that this candidate substances is to can be used for increasing the potential material of insulin sensitivity。
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