CN103239736B - A kind of regulate glycolysis and the method for glyconeogenesis and purposes - Google Patents

Abstract

The invention belongs to biological technical field, be specifically related to a kind of regulate glycolysis and the method for glyconeogenesis and purposes.The method, by regulating the phosphorylation carbohydrate metabolism key enzyme of Fas associated death domain protein, effectively facilitates liver glycolysis, suppression liver glyconeogenesis, thus reduces blood glucose, it is possible to for the screening of carbohydrate metabolism medicine and prepare carbohydrate metabolism medicine.

Description

A kind of regulate glycolysis and the method for glyconeogenesis and purposes

Technical field

The invention belongs to biological technical field, be specifically related to a kind of regulate glycolysis and the method for glyconeogenesis and purposes.

Background technology

Glucose is internal required nutrient, provides main energy source, therefore, stablizing of blood sugar level for cell Particularly significant, carbohydrate metabolism is unbalance, and meeting causes the metabolic disease including diabetes.Internal blood glucose balance is by many factors Common regulation, including consumption and the absorption rate of small intestinal of the saccharide in diet, peripheral tissues's such as muscle, fat are to sugar Absorption, sugar remove in the loss and liver of renal tubules and discharge sugar speed.During these are a series of, liver exists Playing critical effect in glycaemic homeostasis, store sugar by Glycogen synthesis, glycolysis consumption sugar and glycogen degradation and sugar are different A series of activities of raw effect release sugar come balance blood sugar level [Nordlie RC et al., 1999, Annu Rev Nutr 19: 379-406].The glycolysis and the glyconeogenesis process that occur in liver are the regulations by multiple enzyme.Wherein, phosphoenolpyruvate third Keto acid carboxylic kinases (Pepck) and G-6-Pase (G6pase) play the effect of key, and Portugal during glyconeogenesis Glucokinase (Gck) is then the important kinases in glycolytic cycle.

PCK (Pepck) is first rate-limiting enzyme during glyconeogenesis, is catalyzed oxalyl second Acid forms phosphoenolpyruvic acid.Process LAN PCK (Pepck) in liver, mice shows Hyperglycemia, additionally presents hepatic glycogen and MUSCLE GLUCOSE transport protein 4(Glut4) mRNA level in-site reduction etc. is resistance to insulin By relevant symptom, show that process LAN PCK (Pepck) can cause type 2 diabetes mellitus in liver [Valera A et al., 1994, Proc Natl Acad Sci U S A 91:9151-9154].G-6-Pase (G6pc) effect is that catalysis G-6-P is hydrolyzed into glucose, and this is last during glyconeogenesis and glycogen degradation One step, is catalyzed the glucose formed again at the work of glucose transporter 2 (Glut2) through G-6-Pase (G6pc) Liver [van Schaftingen E et al., 2002, Biochem J 362:513-532] is left under with.Glucose-6-phosphorus Acid enzyme (G6pc) plays the effect of key during maintaining euglycemia.In diabetics, due to insulin resistant Or hypoinsulinemia so that the expression of G-6-Pase (G6pc) gene raise [van de Werve G et al., 2000, Eur J Biochem 267:1533-1549].Glucose is transported into by glucose transporter 2 (Glut2) First metabolic activity carried out after liver is phosphorylated into glucose-6-phosphorus exactly under the catalysis of glucokinase (Gck) Acid, G-6-P again by a series of movable enter glycolysiss or participate in Glycogen synthesis [Agius L et al., 2008, Biochem J 414:1-18；Wang R et al., 2013, Gene 516:248-254].Glucokinase (Gck) is main liver Dirty middle expression, also has expression in the tissue that other are relevant with blood glucose regulation, can control pancreas as the induction apparatus of glucose The secretion [Matschinsky FM et al., 2006, Diabetes 55:1-12] of island element.Glucokinase (Gck) is at blood glucose Regulation plays the effect of key, the sudden change of human glocose kinases (Gck) gene and young adult-onset diabetes 2 type (MODY2) relevant.The disappearance of glucokinase (Gck) gene in islet cells and hepatocyte all can cause hyperglycemia, causes MODY2 [Postic C et al., 1999, J Biol Chem 274:305-315, Zhang YL et al., 2004, Acta Pharmacol Sin 25:1659-1665]。

Glycogen Developmental and Metabolic Disorder is the major pathogenetic factor of the metabolic syndromes such as diabetes, obesity, non-alcoholic fatty liver disease.High Blood glucose is as the main clinic symptoms of diabetes, and it produces raw to utilization minimizing, the glycogen of glucose with liver and peripheral tissues Become to increase relevant.Therefore, for the key enzyme during carbohydrate metabolism, effectively facilitate glycolytic cycle in liver, improve glucose Consumption, excessive glyconeogenesis in suppression liver, reduce the generation of endogenous glucose, will be the important targeting for the treatment of diabetes One of and treatment is crucial and difficult point.

FADD(Fas-associated death domain protein, Fas associated death domain protein) be The research groups such as Chinnaiyan, Boldin utilize yeast two-hybrid system to screen obtain one in nineteen ninety-five simultaneously can be with The albumen of death receptor Fas cytoplasmic domain specific effect, due to its comprise one with the death domain height of Fas protein with The domain in source, thus it is named as Fas associated death domain protein (Fas-associated death domain Protein, FADD) [Boldin, M.P. et al., 1995, J. Biol. Chem. 270:7795-7798; Chinnaiyan, A.M. et al., 1995, Cell 81:505-512].208 aminoacid of people's FADD gene code, molecular weight is 23kDa, probably Three functional areas can be divided into, including N end about 76 the amino acid whose DED(death effect domain relevant to apoptosis, Death Effector Domain) and the amino acid whose DD(death domain of 70, C end, death domain), and send out in non-apoptosis Wave about 20 amino acid whose C-terminal fragments of effect.The phosphorylation of the C-terminal fragment of FADD contains a phosphorylation site [Hua ZC et al., 2003, the Immunity 18:513-521] that (194 serines of people, 191 serines of Mus) cause.As Intracellular adaptor protein, FADD can mediate the apoptosis pathway of multiple death receptor induction, in various kinds of cell system The generation that middle energy is apoptosis-induced, is a classical apoptosis signaling molecule.Apoptosis pathway in death receptor induction In, dead signal (such as Fas L) and death receptor (such as Fas) combine, cause the death domain of death receptor (such as Fas) intracellular Form trimer, thus recruit FADD protein by combining the death domain (DD) of FADD, and cause FADD conformational change, Making its DED multimerization, and then recruit Cysteine proteases 8(caspase 8) precursor gathers, and forms dead inducing signaling complex (death-inducing signaling complex, DISC), by producing activated caspase 8, thus excites one Series of downstream caspase cascade reaction, induces apoptosis.

FADD, as the connection albumen of apoptotic signal approach, plays very important work in the generation evolution of tumor With [Zhao Dan, 2006, Interaational magazine 29:152-156；Tourneur, L. et al., 2003, Oncogene 22:2795- 2804].The disappearance of FADD expression and generation closely related [Shin, E.C. et al., 1998, the Cancer Lett. of hepatocarcinoma 134:155-162；Sun, B. et al., 2000, Zhong Hua Gan Zang Bing Za Zhi 8:37-39].FADD egg White expression intensity is closely related with HCC apoptosis, and FADD expressing quantity is the highest, and its apoptosis degree is the most of a relatively high [Lu Dongdong et al., 2002, clinical liver-gallbladder disease magazine, 18:375-376].Find in cancerous cell in many patients with renal cell carcinoma bodies FADD expression substantially reduces [Xu, H. et al., 2009, Cancer Invest, online edition: 2009 Jun 25:1].FADD table Reach abnormal also can play an important role in nonsmall-cell lung cancer [Shin, M.S. et al., 2002, Oncogene 21:4129- 4136]。

For FADD linking in apoptosis and function served as bridge and its relation with multiple disease, existing scholar opens Begin to attempt utilizing FADD that the biological nature of mediating apoptosis can be induced to carry out the treatment of disease.Research finds, FADD mistake Expression can promote the apoptosis of pernicious Glial cells oncocyte.Tumor cell, the glue of 85% is entered by transient transfection FADD gene Matter oncocyte generation apoptosis [Kondo, S. et al., 1998, Human Gene Therapy 9:1599-1608].Utilize Retroviral vector stable transfection tumor cell containing FADD gene, can suppress tumor cell effectively with external in vivo Survival.Separately there are some researches show that the expression of FADD gene can induce the apoptosis [Liu great Qing of Mucoepidermoid carcinoma cell effectively Et al., 2002, Oral and Maxillofacial Surgery magazine, 12:317-320].Except also find, the process LAN of FADD can strengthen tumor cell To drug-induced cell death and the sensitivity of cytotoxicity, reach preferably to treat [Mishima, K. et al., 2003, Int. J. Cancer 105:593-600；Micheau, O. et al., 1999, J. Biol. Chem. 274:7987 -7992；Yin, A. et al., Med. Oncol. online edition: 2009 May 5].

FADD is except having important application prospect in addition in oncotherapy, and research shows that the apoptosis that Fas mediates is class The one of the main reasons of rheumatic arthritis patient articular synovial cell's degeneration, wherein the signal conduction of FADD-caspase-8 Approach play a significant role during the apoptosis of synoviocytes of Patients With Rheumatoid Arthritis [Kobayashi, T. et al., 1999, Curr. Opin. Rheumatol. 11:188-193].Utilize Adenovirus Transfection that FADD gene proceeds to rheumatoid to close After the synovial cell that joint is scorching, the apoptosis of the synovial cell causing pathological changes because of hyper-proliferative can be caused.It addition, in the class of immunodeficiency Local injection adenovirus-FADD(Ad-FADD in rheumatic arthritis model mice) after can remove the synovial cell of pathological changes, and this Effect is confined to synovial tissue, and does not cause neighbouring articular chondrocyte apoptosis.Thus FADD process LAN can effectively treat class wind Wet arthritis [Kobayashi, T et al., 2000, Gene Therapy 7:527-533].

Summary of the invention

It is an object of the invention to provide a kind of method regulating glycolysis and glyconeogenesis, it is characterised in that: express simulation Fas The gene of associated death domain protein phosphorylation, or with Fas associated death domain protein phosphorylation degree for accusing of sieve Choosing improves the material of Fas associated death domain protein phosphorylation, and the common purpose of said method is to improve Fas associated death knot The phosphorylation of structure territory albumen.

Further, a kind of method regulating glycolysis and glyconeogenesis, it is characterised in that: regulation reduces phosphoenolpyruvate third Keto acid carboxylic kinases and G-6-Pase activity, improve glucokinase.

Further, a kind of method answering in preparation treatment carbohydrate metabolism disease medicament regulating glycolysis and glyconeogenesis With.

Further, the method for above-mentioned regulation glycolysis and glyconeogenesis answering in preparation treatment carbohydrate metabolism disease medicament With, with existing carbohydrate metabolism disease therapeuticing medicine and the use in conjunction of technology.

Further, a kind of pharmaceutical composition, it is characterised in that: around a kind of regulate glycolysis and the method for glyconeogenesis, Utilize the combination formula that pharmaceutically conventional carrier or excipient or diluent are carried out.

Further, a kind of method regulating glycolysis and glyconeogenesis is preparing the application treated in diabetes medicament.

In a first aspect of the present invention, it is provided that a kind of method regulating glycolysis and glyconeogenesis, it is characterised in that pass through The phosphorylation of regulation Fas associated death domain protein, thus realize glycolysis and the regulation of glyconeogenesis.

Described regulation glycolysis and the method for glyconeogenesis, can not only reduce PCK and Fructus Vitis viniferae Sugar-6-phosphatase activity, and glucokinase can be improved.

In a second aspect of the present invention, it is provided that a kind of by screening can improve Fas associated death domain protein The method of the material of phosphorylation, it is characterized in that with Fas associated death domain protein phosphorylation degree be changed to accuse of screening The material of Fas associated death domain protein phosphorylation can be improved, thus realize glycolysis and the regulation of glyconeogenesis.

The described material that can improve Fas associated death domain protein phosphorylation, include can improve Fas be correlated with The macromolecular substances of death domain protein phosphorylation level, such as, protein, nucleic acid, polysaccharide, fatty acid, vitamin and Nano molecular etc.；Also include the small-molecule substance that can improve Fas associated death domain protein phosphorylation level, such as, The product of natural product, chemosynthesis or chemical improvement, organic molecule, inorganic molecule etc..

In a third aspect of the present invention, it is provided that a kind of by introducing simulation Fas associated death domain protein phosphorylation Gene express, thus the method regulating glycolysis and glyconeogenesis.

The gene of described simulation Fas associated death domain protein phosphorylation, is characterized in that referring to people's Fas associated death knot People's Fas associated death domain protein that 194th mutant serine is aspartic acid or glutamic acid sudden change of structure territory albumen Gene.The gene of above-mentioned simulation Fas associated death domain protein phosphorylation can be used for gene therapy.

In a fourth aspect of the present invention, it is provided that the method for regulation glycolysis described above and glyconeogenesis is preparing carbohydrate metabolism Application in medicine.Described application includes can improve Fas associated death domain protein by what screening obtained The material of phosphorylation or the gene of simulation Fas associated death domain protein phosphorylation are individually used for preparing carbohydrate metabolism curative Thing, or be grouped compound with existing carbohydrate metabolism medicine, or with existing chemotherapy, treatment by Chinese herbs, Biotherapeutics, The methods such as naturopathy are conjunctive use in carbohydrate metabolism is treated.

In a fifth aspect of the present invention, it is provided that a kind of pharmaceutical composition, including pharmaceutically acceptable carrier or figuration Agent or diluent, and the regulation glycolysis and the method for glyconeogenesis described in the claim 1 of effective dose.

In a sixth aspect of the present invention, it is provided that the method for regulation glycolysis described above and glyconeogenesis is in preparation diabetes Concrete application in medicine.

Comparing compared with carbohydrate metabolism disease method, the present invention has a following beneficial effect:

(1) achieve by regulating and controlling carbohydrate metabolism while liver glycolysis and glyconeogenesis: the most conventional tune Save glycometabolic method be typically all regulation or directly increase the level of insulin, or the sensitivity that regulation is to insulin, Play the effect of regulation blood glucose.This patent is then effectively to reduce blood glucose by regulating liver glycolysis and glyconeogenesis simultaneously Level.

(2) achieve first by the regulation of Fas associated death domain protein phosphorylation is regulated and controled carbohydrate metabolism: this Invention first passage realizes realizing the phosphorylated regulation of a classical apoptosis protein to glycometabolic regulation, thus Effectively reduce blood sugar level.

(3) achieve first to regulate and control while liver glycolysis and glyconeogenesis: glycolysis and glyconeogenesis be two different Process.The present invention by the regulation of Fas associated death domain protein phosphorylation is achieved notable lower in liver with sugar The closely-related G-6-Pase of heteroplasia (G6pc) and the expression of PCK (Pepck), from And reduce the generation of glucose；The present invention achieves further through to the regulation of Fas associated death domain protein phosphorylation simultaneously Notable rise participates in glucolytic key enzyme--the expression of glucokinase (Gck), improves the utilization of sugar, eventually reduces Blood sugar level.

(4) achieve first liver glycolysis and the coordinated regulation of multiple key enzymes of glyconeogenesis: the present invention is by right The regulation of Fas associated death domain protein phosphorylation achieve protein kinase B (Akt) phosphorylation and downstream protein transcription thereof because of The regulation and control of sub-Foxo1 phosphorylation, have adjusted the G-6-Pase (G6pc) in transcription factor Foxo1 downstream, phosphoric acid alkene then The expression of alcohol of formula pyruvate carboxykinase (Pepck).The present invention is by Fructus Vitis viniferae closely-related with glyconeogenesis in final regulation liver Sugar-6-phosphatase (G6pc), PCK (Pepck), glycolysis key enzyme--glucokinase (Gck) Coordinated regulation, thus significantly significantly reduce blood sugar level.

(5) a kind of new screening regulation glycolysis and glyconeogenesis medicine or the method for Therapeutic Method are provided: the present invention Provide a kind of phosphorylation by regulation and control Fas associated death domain protein to realize regulating glycolysis and the side of glyconeogenesis Method, utilizes the phosphorylation of Fas associated death domain protein to do index, and those skilled in the art can utilize the most conventional inspection The screening of survey method can regulate the material of the phosphorylation of Fas associated death domain protein, including protein, nucleic acid, polysaccharide, fat The macromolecular substances such as fat acid, vitamin and nano molecular thereof, also include the product of natural product, chemosynthesis or chemical improvement The small-molecule substances such as thing, organic molecule, non-polar molecule.

Accompanying drawing explanation

It is clearly understood to make present disclosure be easier to, below according to specific embodiment and combine accompanying drawing, right The present invention is described in further detail, wherein

Fig. 1. FADD phosphorylation is on the impact of associated hormone level in blood sugar level and serum.

Figure 1A. the blood sugar content of mice under fasting and normal condition: the 1. normal control mice under fasting state；2. FADD phosphorylation mice under fasting state；3. the control mice under normal condition；4. the FADD phosphorylation under normal condition Mice.

Figure 1B. the insulin level in mice serum under fasting state: 1. normal control mice；2. FADD phosphorylation Mice；

Glucose-dependent insulinotropic peptide (GIP) level in mice serum under Fig. 1 C. fasting state: 1. normal control is little Mus；2. FADD phosphorylation mice；

Glucagon-like-peptide-1 (GLP-1) level in mice serum under Fig. 1 D. fasting state: 1. normal control is little Mus；2. FADD phosphorylation mice.6-13 mice is often organized) according to being expressed as meansigma methods ± SEM(.Collect the blood of 9-11 week mice Carry out biochemical analysis.Significance analysis, * P < 0.05 is carried out by t-test.

Fig. 2. FADD phosphorylation is on mice sugar tolerance and the impact of insulin sensitivity.

Fig. 2 A. glucose tolerance test.Mice is by the dosage lumbar injection glucose of 1.0 g/kg, every the specific time Interval test glucose level (often 3 mices of group), white diamond represents that control mice, black square represent that FADD phosphorylation is little Mus；

In Fig. 2 B. glucose tolerance test, Area under the curve of blood glucose: 1. normal control mice；2. FADD phosphorylation is little Mus；

Fig. 2 C. insulin sensitivity is tested.Mice is by the dosage lumbar injection insulin of 0.5 U/kg, every specifically Time interval test glucose level (often 3 mices of group), white diamond represents that control mice, black square represent FADD phosphoric acid Change mice；

In the experiment of Fig. 2 D. insulin sensitivity, the total amount that after injection of insulin, glucose reduces: 1. normal control mice； 2. FADD phosphorylation mice；Data are all expressed as meansigma methods ± SEM.Significance analysis is carried out by t-test, * P < 0.05, * * P < 0.01, * * * P < 0.001.

Fig. 3. the impact on the expression of carbohydrate metabolism associated protein of the FADD phosphorylation.

The Western blot result of carbohydrate metabolism key enzyme in Fig. 3 A. mouse liver, including Pepck, G6pc and Gck, with Tublin is as internal standard: 1. normal control mice；2. FADD phosphorylation mice；

The mrna expression level of carbohydrate metabolism related gene: 1.G6pc in Fig. 3 B. mouse liver；2.Pepck；3.Gck； 4.Foxo1；Wherein, white is normal control mice, and black is FADD phosphorylation mice.Often 4-6 mice of group, each sample three Individual repetition, with GAPDH as internal standard；

After Fig. 3 C. insulin processes, Western Blot is in the liver sample preparation taking mice, detects pAkt, Akt and GAPDH Protein content, right figure is to the quantitative analysis of pAkt in Western Blot result: 1. the comparison before insulin stimulating is little Mus；2. the control mice after insulin stimulating；3. the FADD phosphorylation mice before insulin stimulating；4. after insulin stimulating FADD phosphorylation mice；

The immunofluorescence of Fig. 3 D. mouse liver section Foxo1, Hoechst dye core (often 3 mices of group), scale=20 m；Data are all expressed as meansigma methods ± SEM.Significance analysis is carried out by t-test, * P < 0.05, * * P < 0.01, * * * P < 0.001。

Detailed description of the invention

Embodiment 1

Fas associated death domain protein (FADD) phosphorylation is on the impact of associated hormone level in blood sugar level and serum

All of experiment mice is all aseptic in ventilation, carries out captive breeding under the SPF rank environment of constant temperature and humidity.Little Mus can freely obtain water and feedstuff.The breeding of FADD phosphorylation mice carries out [Hua ZC according to the method for reported in literature before Et al., 2003, Immunity 18:, 513--521].Gene identification is carried out by PCR method.

Mice overnight starvation.Blood sugar content is detected by blood glucose meter (Roche, Mannheim, Germany).Blood Insulin content Rat/Mouse Insulin ELISA kit (Millipore, Billerica, MA, USA) in clear Detect.Glucose-dependent insulinotropic peptide (GIP) in serum, glucagon-like-peptide-1 (GLP-1) are horizontally through outstanding Wick-containing sheet carries out detecting (Bio-Rad Laboratories, Hercules, CA, USA).

Owing to, under normal condition, FADD is based on non-phosphorylating, and phosphorylation form is only in specific physiology and pathological state Lower just existence.In order to observe FADD phosphorylation to glycometabolic impact, this patent constructs the gene of simulation FADD phosphorylation and changes Make mice (to be aspartic acid by 191 mutant serines of murine FADD genes, simulate the FADD of phosphorylation, this kind of model with this It has been internationally recognized) [Hua ZC et al., 2003, Immunity 18:513-521].Blood glucose to FADD phosphorylation mice (glucose) testing result shows, the blood sugar level of FADD phosphorylation mice is the most still in fasting Under the conditions of be all significantly lower than its littermate control mice, its blood sugar level is about the 2/3 of control mice, as shown in Fig. 1 A.Blood glucose Balance is to be regulated by hormonal readiness to a certain extent, and in pancreas, the insulin (insulin) of secretion is in the regulation of blood glucose Playing the effect of key, therefore, this patent also have detected the content of serum insulin, and Fig. 1 B shows that FADD phosphorylation is little The content of Mus serum insulin is higher than control mice.Additionally, this patent also to have detected in serum other relevant to diabetes The level of hormone.Glucose-dependent insulinotropic peptide (GIP) and glucagon-like-peptide-1 (GLP-1) are FADD phosphorylation mice In also raise (Fig. 1 C and 1D), they can promote the secretion of insulin, with it is observed that serum in hyperinsulinism water Flat consistent.Therefore, the insulinotropic peptide (GIP) that in FADD phosphorylation mice, insulin level raises, promotes insulin to raise Also raise with glucagon-like-peptide-1 (GLP-1) level, thus cause blood glucose to reduce.

Embodiment 2

FADD phosphorylation is on mice sugar tolerance and the impact of insulin sensitivity

In glucose tolerance test, mice overnight starvation pneumoretroperitoneum injectable dextrose monohydrate (1.0 g/kg), in injection after 0, 15, blood sugar content is surveyed after 30,60 and 90 minutes.In insulin resistant is tested, mice is after hungry 6 hours, lumbar injection islets of langerhans Element (0.5 U/kg).Blood sugar content is surveyed after 0,15,30,60 and 90 minutes after injection.

In order in further Study Mouse, FADD phosphorylation has carried out abdominal cavity note for the impact of glucose homeostasis, the present invention The glucose tolerance test (IPGTT) penetrated and insulin resistant experiment (IPITT).In glucose tolerance test (IPGTT), The blood sugar level of FADD phosphorylation mice substantially reduces a lot (Fig. 2 A) than control mice.Glucose post-stimulatory blood glucose liter High Area under Curve data shows, FADD phosphorylation mice area under curve is only 2/3(Fig. 2 B of normal control mice). This shows that the Scavenging activity of glucose is strengthened under glucose stimulates by FADD phosphorylation mice.

The present invention has also carried out insulin resistant experiment (IPITT) of lumbar injection, exists detecting the sensitivity of insulin Whether FADD phosphorylation mice changes.After injection of insulin, although the blood glucose of FADD phosphorylation mice drops to comparison According to mice much lower (Fig. 2 C), but they relative blood glucose decline change approximation (Fig. 2 D) of level, show FADD phosphoric acid The insulin sensitivity change changing mice is the most notable.

Embodiment 3

The impact on the expression of carbohydrate metabolism associated protein of the FADD phosphorylation

Immunofluorescence: the liver taken out from animal body is fixed in the phosphoric acid buffer formalin of 10%, paraffin embedding After, it is cut into the thin slice of 5 m thickness, with the antibody of anti-Foxo1 as the Foxo1 in an anti-location liver, uses Alexa Fluor 488-conjugated anti-rabbit IgG antibody resists as two, and Hoechst contaminates core.Fluorescence microscope is carried out Observe.

Western Blot: mice overnight starvation pneumoretroperitoneum injection 1U/kg insulin.Put to death after 15 minutes and take liver Dirty.It is organized in cell pyrolysis liquid (50 mM Tris-HCl, pH 7.4,250 mM NaCl, 50 mM NaF, 5 mM EDTA, 5 mM phosphoglycerols, 1 mM Na3VO4,1% NP40) in after homogenate 12,000 rpm be centrifuged, collect supernatant, And detect protein concentration by Bradford method.Equivalent (about 50 g) albumen turns after being separated by electrophoresis with the PAGE gel of 12% Move on in pvdf membrane.Examine with Akt, phospho-Akt (Ser 473), the antibody of G6pc, Pepck, Gck the most respectively Test.

RNA extracts and the most real-time PCR: by TRIzol (Invitrogen, Carlsbad, CA, USA) process group Extract RNA according to subsidiary description after knitting, then with PrimeScript RT reagent Kit (Takara, Otsu, Shiga, Japan) carry out reverse transcription and prepare cDNA.PCR(qRT-PCR the most in real time) at ABI machine (Applied Biosystems, Foster City. CA, USA) on carry out.Primer is as follows: mG6pc: forward primer: 5 '- TCGGAGACTGGTTCAACCTC-3’；Reverse primer: 5 '-ACAGGTGACAGGGAACTGCT-3 '；MGck: forward primer: 5 '- GAGGTCGGCATGATTGTGGGCA-3’；Reverse primer: 5 '-ACACACATGC GCCCCTCATCGCC-3 '；MPepck1: just To primer: 5 '-CTAACTTGGCCATGATGAACC-3 '；Reverse primer: 5 '-CTTCACTGAGGTGCCAGGAG-3 '； MFoxo1: forward primer: 5 '-TGTCAGGCT AAGAGTTAGTGAGCA-3 '；Reverse primer: 5 '- GGGTGAAGGGCATCTTTG-3’；MGAPDH: forward primer: 5 '-ACTGAGGACCAGGTTGTC-3 '；Reverse primer: 5 '- TGCTGTAGCCGTATT CATTG-3’；.All of result is all the meansigma methods of 3 experiments, using GAPDH as internal reference.

Data analysis: all of data result is all expressed as meansigma methods ± SEM.Use Prism software Two-tailed Student ' s t-test in (GraphPad, San Diego, CA) is carried out between two kinds of genotype Difference analysis.It is considered data when P value is less than 0.05 and there is significant difference.

Liver is glycometabolic main place, and the balance of blood glucose is played pivotal role.Therefore, the present invention have detected liver Middle involved in sugar heteroplasia and the key protein of glycolytic cycle, whether the expression observing these albumen is affected by FADD phosphorylation. Result shows, G-6-Pase (G6pc), PCK (Pepck) and glucokinase (Gck) Albumen and the mRNA level in-site of the these three glycometabolic enzyme of participation all there occurs significant change (figure in FADD phosphorylation mice 3A, 3B).Speed limit protein glucose-6-phosphatase (G6pc) during glyconeogenesis and PCK (Pepck) all lower, reduce the generation of source property glucose；And the increase that glucokinase (Gck) is expressed, then accelerate sugar The process of zymolysis, promotes the utilization of internal glucose.

For the hypoglycemic activity of further investigated FADD phosphorylation, the present invention observes and plays key work in blood glucose balance Insulin signaling pathway.Protein kinase B (Akt) phosphorylation journey under quiescent condition, in FADD phosphorylation mouse liver Degree rises (Fig. 3 C).After insulin stimulating, the phosphorylation degree of protein kinase B (Akt) is still gone up in FADD phosphorylation mice Adjust.But by the quantitative analysis to Western bolt result, it has been found that, although after insulin stimulating, FADD phosphorylation The phosphorylation degree of murine protein kinase b (Akt) remains above normal control mice (Fig. 3 C), but relative to substrate level Upper modulation, FADD phosphorylation is not significantly different from control mice, and the upper modulation of contrary FADD phosphorylation mice is slightly below Control mice.This shows that the sensitivity of insulin is not changed in by FADD phosphorylation mice, and this is real with insulin resistant before The result tested is consistent (Fig. 2).

The present invention also have detected the downstream protein transcription factor Foxo1 of protein kinase B (Akt).Turning of unphosphorylated form Record factor Foxo1 is typically expressed in nucleus, and transcription factor Foxo1 of the phosphorylation after being activated by protein kinase B (Akt) goes out Core.Transcription factor Foxo1 of process LAN phosphorylation can significantly reduce blood sugar level.The transcription factor of liver tissue slices The immunofluorescence dyeing result of Foxo1 shows: transcription factor Foxo1 the most all goes out core；And in normal control mice, transcribe Factor Foxo1 major part is all distributed (Fig. 3 D) in core.This shows that the phosphorylation of FADD can improve protein kinase B (Akt) and live Property, and then cause the phosphorylation level of transcription factor Foxo1 also to raise.And Foxo1 is as transcription factor, Fructus Vitis viniferae can be promoted Sugar-6-phosphatase (G6pc) and the expression of PCK (Pepck) gene.In the present invention, transcribe because of Core is gone out, it is suppressed that G-6-Pase (G6pc) and PCK after sub-Foxo1 phosphorylation (Pepck) albumen of the two gene observed in expression, this result and Fig. 3 A and 3B of the present invention and mRNA level in-site fall Low completely the same.

The phosphorylation of FADD can affect G-6-Pase (G6pc), phosphoenolpyruvate in involved in sugar metabolic process Pyruvate carboxykinase (Pepck1), the expression of glucokinase (Gck) these key enzymes, thus promote the glycolysis profit to sugar With, the generation of the glucose that suppression glyconeogenesis causes, the final body Scavenging activity to glucose that improves, reduction blood sugar level. FADD phosphorylation is relevant with the rise of protein kinase B (Akt) phosphorylation level to a certain extent on the impact of blood glucose balance.Cause This, the carbohydrate metabolism disease that FADD phosphoric acid of the present invention turns to treat including diabetes provides a new drug target.Profit It is obtained with regulating and controlling the material of FADD phosphorylation with FADD phosphorylation level as screening mark, thus finds regulation sugar The medicine of metabolism.

The present invention, by proceeding to the FADD mutant gene of a simulation FADD phosphorylation, is directly realized glucose-6- Phosphatase (G6pc), PCK (Pepck1), the expression of glucokinase (Gck) these key enzymes Regulation and control, thus effectively have adjusted the carbohydrate metabolism including blood sugar level.Therefore, the FADD sudden change base of simulation FADD phosphorylation The gene therapy of cause necessarily becomes a kind of effective means of regulation carbohydrate metabolism disease.

Therefore, carbohydrate metabolism disease can be directly applied to by the method for FADD phosphorylated regulation glycolysis and glyconeogenesis control Treat the discovery of medicine, preparation, and treatment.The method and can with existing chemotherapy, treatment by Chinese herbs, Biotherapeutics, The carbohydrate metabolism Therapeutic Method conjunctive use such as naturopathy, produce more preferable therapeutic effect.

The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually It is incorporated as with reference to equally.In addition, it is to be understood that after having read the appeal teachings of the present invention, those skilled in the art are permissible To the present invention, various modifications may be made or changes, and these equivalent form of values should be included within the scope of the present invention.

Claims (6)

1. one kind is passed through the Fas glycometabolic method of associated death domain protein phosphorylated regulation, it is characterised in that express simulation The gene of Fas associated death domain protein phosphorylation, or by Fas associated death domain protein phosphorylation can be improved Compound, improve Fas associated death domain protein phosphorylation regulate carbohydrate metabolism, described method shall not be applied to disease Treatment.

One the most according to claim 1 passes through the Fas glycometabolic method of associated death domain protein phosphorylated regulation Application in regulation carbohydrate metabolism, described application is not used in the treatment of disease.

One the most according to claim 1 passes through the Fas glycometabolic method of associated death domain protein phosphorylated regulation Application at regulation glucokinase, it is characterised in that adjusted by regulation Fas associated death domain protein phosphorylation Joint reduces PCK and G-6-Pase activity, improves glucokinase, described application It is not used in the treatment of disease.

One the most according to claim 1 passes through the Fas glycometabolic method of associated death domain protein phosphorylated regulation Application in regulation glycolysis and glyconeogenesis, described application is not used in the treatment of disease.

5. the one described in claim 1 regulates carbohydrate metabolism medicine as target spot in preparation by Fas associated death domain protein Application in thing.

One the most according to claim 5 regulates sugar generation as target spot in preparation by Fas associated death domain protein Thank to the application in medicine, it is characterized in that and existing carbohydrate metabolism medicine and the use in conjunction of technology.