CN101955981A - A kind of novel targets and biological characteristics thereof that is used for diabetes, obesity and cardiovascular disease therapies drug development - Google Patents

Abstract

The invention discloses acyltransferase ALCAT1 as a kind of target spot that is used for diabetes, obesity and cardiovascular disease therapies drug development.The present invention has pointed out under oxidative stress condition ALCAT1 to cause the pathology reconstruct effect of diphosphatidylglycerol, thereby cause the symptom fat relevant such as mitochondria dysfunction and insulin resistant with diet induced, ALCAT1 can be used as a kind of oxidative stress and the regulatory gene of mitochondria dysfunction in metabolic disease and aging, causes that in the active oxygen increase diphosphatidylglycerol conformational change raises ALCAT1 and causes the leakage of plastosome proton, oxidative stress and mitochondria dysfunction further to aggravate insulin resistant and metabolic deficiency.Therefore, the chemical reagent that has suppressed ALCAT1 can be obesity and the associated metabolic complication provides new methods of treatment.

Description

A kind of novel targets and biological characteristics thereof that is used for diabetes, obesity and cardiovascular disease therapies drug development

Technical field

The present invention relates to ALCAT1 (front center phosphoacylase) change of diphosphatidylglycerol chlorine conformation is caused the vital role that plays in the process of diet induced obesity at mitochondria dysfunction, insulin resistant, is the novel targets of drug treatment of diabetic, obesity and cardiovascular diseases.

Background technology

Mitochondria dysfunction is confirmed as a kind of common metabolic obesity complication recently.Yet these mitochondria activities are too high relevant with Asia Indian's severe insulin resistance disease.The oxidative phosphorylation reduced activity can prevent diet induced in the mouse of AIF (plastosome flavoprotein apoptosis inducing factor) disappearance obesity and insulin resistant disease.In addition, all euglycemic agents all have the activity that suppresses the plastosome composite I comprising most popular antidiabetic medicine thiazole and N1,N1-Dimethylbiguanide class.Therefore, the pathogenic effects molecular mechanism that causes fat of mitochondria dysfunction still remains clarification.

Cumulative evidence shows, a lot of active oxygens are relevant with disease of metabolism and aging to the mitochondria dysfunction that the damage of diphosphatidylglycerol causes.Diphosphatidylglycerol is a kind of polysaccharide phosphatide, for keeping the keying action that plastosome biological function and mitochondrial protein activity have.Metabolism in the great majority tissue, the side chain of diphosphatidylglycerol mainly be linolic acid domination, linolic acid is requisite in supporting its mitochondrial function.Yet this unique acyl group is modified the synthetic (rather than reinventing) length by length that starts anew, and relates to desacylation-reacylation cycle or different phosphatide up time acylation.Diphosphatidylglycerol is reinvented the process that is considered to repair oxidative damage, because the active oxygen of diphosphatidylglycerol responsive especially (its pair key is easy to oxidized near the mitochondrial inner membrane that produces active oxygen), active oxygen increases suddenly, just may cause diphosphatidylglycerol oxidation and quantity not sufficient, and then cause mitochondria dysfunction.Non-oxide diphosphatidylglycerol is active essential by plastosome bio-energy and mitochondrial protein.Therefore, diphosphatidylglycerol is reinvented and is confirmed as diabetes, obesity, cardiovascular disorder and old and feeble common defects, and is that the diphosphatidylglycerol acyl group constitutes the sign that changes.Yet, reinvent the molecular mechanism of the obesity morbidity that the food habits with mitochondria dysfunction and insulin resistant disease causes for diphosphatidylglycerol and also know little about it.

ALCAT1 be first found can catalysis before Val (lysoCL) to the transferring enzyme of diphosphatidylglycerol acyl group, be the committed step that diphosphatidylglycerol is reinvented.The tetrahydrochysene diphosphatidylglycerol is all not enough in mammiferous cell, ALCAT1 up-regulated in the patient's of hyperthyroidism heart and the liver, and this shows it is the process that pathological effect has caused diphosphatidylglycerol to be reinvented.

Summary of the invention

The objective of the invention is to find a kind of novel targets that is used for diabetes, obesity and cardiovascular disease therapies drug development.

From the yeast to the Mammals, diphosphatidylglycerol has vital role in regulating mitochondrial function and oxidative stress process, is made up of its 4 chain acyls to mediate.In the insulin sensitivity tissue, diphosphatidylglycerol acyl chain is that the linolic acid by its best biological function is main decision.The function of this uniqueness also is embodied in the extremely sensitive oxidative stress infringement of the diphosphatidylglycerol compensatory process; phosphatide and acyltransferase have remedied the damage that oxidation caused of the active oxygen acyl chain of lipid acid in this process, have prevented that obstacle from appearring in mitochondrial function.Therefore, reinvent the pathology diphosphatidylglycerol and just become a mitochondria dysfunction that causes by the raising of the reactive oxygen species pathologic, physiologic characteristics of (comprising diabetes, obesity, cardiac failure and aging).But, diphosphatidylglycerol pathology reinvent and with and the molecular mechanism of the mitochondria dysfunction that causes still unknown.

Utilize cell and the ALCAT1 knock out mice model for the basis, we find the effect of ALCAT1 in pathologic diphosphatidylglycerol remodeling process for the first time, also can be observed in the pathologic process of diabetes and obesity.Therefore, because making the method for ALCAT1 mouse gene inactivation targetedly, caused oxidative stress of ALCAT1 overexpression and mitochondria dysfunction can protect mouse to avoid suffering to the mammiferous cell of being cultivated: to comprise mitochondria dysfunction, insulin resistant, lipid peroxidation and liver cirrhosis because obesity and the associated metabolic complication thereof that hyperalimentation causes.

Diphosphatidylglycerol is not enough and the change diphosphatidylglycerol acyl group is the common feature of diabetes and obesity; pathologic diphosphatidylglycerol is reinvented the C16 content that shows as C18 in the diphosphatidylglycerol and is reduced; comprise in four inferior oily acyloxy Val-diphosphatidylglycerols the most general a kind of, and the increasing of the content of DHA (docosahexenoic acid).ALCAT1 knocks out and has improved in the heart four inferior oily acyloxy Val-diphosphatidylglycerols greatly and reduced DHA-diphosphatidylglycerol level in liver targetedly, and the result is with aforementioned consistent, and the Hela cell of ALCAT1 overexpression lacks linolic acid.Our result of study has confirmed that further high-fat diet causes four inferior oily acyloxy Val-diphosphatidylglycerols to lack at rat heart.

It is that oxidative pressure causes mitochondria dysfunction and metabolism complication that the research metabolic disease has one of significant problem to be solved, as insulin resistant.Our work has supported that ALCAT1 causes the pathology reconstruct effect of diphosphatidylglycerol under the oxidative stress condition, thereby causes the symptom fat relevant with diet induced such as mitochondria dysfunction and insulin resistant.At first, ALCAT1 overexpression defective causes diphosphatidylglycerol to reduce and DHA increases, and has been proved to be the mammalian cell apoptosis that can cause oxidative stress, lipid peroxidation and cultivation.Polyunsaturated fatty acid in diphosphatidylglycerol (PUFA) content increases and can be regarded as the snperoxiaized index of diphosphatidylglycerol, and it can further aggravate response to oxidative stress.Next influences the reaction 4 of oxidation-respiration chain (reaction of this step is a main source of producing active oxygen) thereby the ALCAT1 overexpression has caused proton leakage and UCP3 up-regulated.The mitochondrial respiratory uncoupling is confirmed as the common metabolic deficiency recently, can be in diabetes, and obesity, hyperthyroid mouse heart separates in the plastosome that obtains to be observed.The 3rd, the ALCAT1 overexpression increases active oxygen production greatly in the C2C12 cell, the reaction when this has also further aggravated the hydrogen peroxide stimulation.Consistent with the increase of oxidative stress level, several down regulation of gene expression of coding antioxidase, and those stimulate the gene of oxidative stresss to raise.In these genes that raised by ALCAT1, Nox4 expresses increases by 28 times.Nox4 has the oxidasic homology of NAD (P) H, and active oxygen stimulates generation in the insulin sensitivity tissue.Nox4 is proved to be recently and is positioned at plastosome, and is the main source of diabetic animal active oxygen.The 4th, prove that further ALCAT1 is the reason of oxidative stress pathogenic effects, feed the ALCAT1-of food rich in fat/-the mouse lipid obviously reduces in the peroxidation of liver and heart.At last, ALCAT1 mRNA up-regulated in hyperfunction mouse liver of Tiroidina and heart, Tiroidina is hyperfunction can to stimulate diphosphatidylglycerol peroxidation and oxidative stress.

We influence diphosphatidylglycerol at ALCAT1 and have found a new molecular mechanism, the mitochondria dysfunction that insulin resistant causes in reinventing.Research in the past thinks that the plastosome oxygenizement is impaired in diabetes and accurate diabetics.But this theory has been challenged in nearest report, and mitochondria activity strengthens relevant with American Indians' severe insulin resistance, and is also relevant with rodent diet inductive obesity.Increase in the test with insulin resistant supporting ALCAT1 to regulate mitochondria activity, the production rate of ATP rises and produces insulin resistant in the C2C12 of ALCAT1 overexpression cell, ALCAT1-that liver and heart ALCAT1 knock out/-mouse in insulin sensitivity strengthen.This meets our observation, and the mitochondrial ATP production rate significantly raises in diabetes db/db mouse, and this is normal fully after adding Rother row ketone (rosiglitazone is the PPAR inhibitor, can increase the susceptibility of Regular Insulin) nursing all around.Regular Insulin has been proved to be can increase ATP production rate in the healthy human body skeletal muscle, but can not act on type ii diabetes patient and their offspring, because they have insulin resistant.Can regulate the insulin sensitivity unanimity with ALCAT1, fully passivation in the C2C12 cell that acts on the ALCAT1 overexpression of insulin stimulating ATP production rate, this with human diabetic patient in observed consistent.ALCAT1 has regulating effect in plastosome increased activity and insulin resistant, isolating plastosome mesocomplex 1 increased activity in the C2C12 cell of ALCAT1 overexpression, isolating plastosome complex body 1 active reduction of ALCAT1 knock-out mice liver.Diphosphatidylglycerol be complex body 1 live type required also be that active oxygen production and diphosphatidylglycerol peroxidation process are necessary.Aging, fatty liver disease, myocardial ischemia-reperfusion pathologic process that our result of study and the former snperoxiaized active oxygen of diphosphatidylglycerol cause in plastosome complex body I dysfunction are consistent.In addition, the euglycemic agent of all known type ii diabetes has been proved to be and can have suppressed plastosome complex body I activity, comprises N1,N1-Dimethylbiguanide, the activator of thiazole and AMPK.In addition, being proved to be as diphenyl iodonium and Opsonin can the lowering blood glucose level, suppresses complicated I activity and oxygen-consumption.

Fat in developed country circle just popular, yet, its basic reason is short in understanding hindered this disease medicament exploitation of effective treatment.Our result of study shows that ALCAT1 has vital role in regulating the fat process of energy balance and diet induced.This viewpoint by ALCAT1-/-mouse has higher basal metabolic rate(BMR) (RMR) and the brown fat increased activity is supported.When normal physiological is moving, basal metabolic rate(BMR) (RMR) account for total energy expenditure 65%.ALCAT1mRNA preferentially expresses in the tissue with higher static metabolic rate (RMR), and this also is the vaild evidence that ALCAT1 regulates the RMR effect.Except resisting the diet induced obesity, ALCAT1-/-mouse also avoids suffering from the relevant disease that comprises with the metabolism complication as obesity, liver cirrhosis etc. because of being protected.This was supported by a former report, this report thinks that carnitine palmitoyltransferase 1 (a kind of enzyme that Fatty Acid Oxidation is required) activity needs diphosphatidylglycerol, the not enough infringement of diphosphatidylglycerol liver fat acid oxidase, and the DHA level that increases is relevant with the seizure of disease of rodent fatty liver with lipid peroxidation.In fact, thus the ALCAT1 deficiency has hindered the probability that the contents level of DHA has reduced concurrent liver cirrhosis.ALCAT1-/-mouse also shows plastosome Fatty Acid Oxidation rate and liver lipid peroxidation, this is relevant with the obesity that diet causes usually.Report before the result has further confirmed: obesity increased lipid peroxidation level and diphosphatidylglycerol peroxidation with late period nonalcoholic fatty liver disease relevant.

Importantly, our result of study has great influence, because pathologic diphosphatidylglycerol conformational change is considered to diabetes, obesity, cardiovascular disorder, aged common defects.Our results suggest acyltransferase the regulation mechanism in metabolic disease and aging of ALCAT1 as a kind of oxidative stress and mitochondria dysfunction.Therefore, thus cause that in the active oxygen increase diphosphatidylglycerol conformational change raises ALCAT1 and causes the mechanism of the leakage of plastosome proton, oxidative stress and mitochondria dysfunction further to aggravate insulin resistant and metabolic deficiency.Therefore, the chemical reagent that has suppressed ALCAT1 can be obesity and the associated metabolic complication provides new methods of treatment.

Description of drawings

Fig. 1 is with the composition of the C2C12 cell diphosphatidylglycerol acyl group composition of anionic electrodeposition spraying mass spectroscopy stable transfection ALCAT1 and empty carrier.From the C2C12 cell extraction lipid of transfection ALCAT1 and empty carrier, with the anionic electrodeposition mass spectroscopy of spraying.The result shows that the ALCAT1 overexpression is C16 and reduction of C18 fatty acid content and the rising of long chain polyunsaturated fatty acids content optionally, and total diphosphatidylglycerol reduces.

Fig. 2 mitochondria dysfunction and oxidative stress are at the C2C12 of ALCAT1 overexpression cell.A is with Clark-type electrode measurement C2C12 cell suspending liquid oxygen depletion speed.Base respiration (control) after the measurement of the cellular respiration of state 4 breathings and maximum, adds the oligomycin of 3uM and the FCCP of 2uM.B, at oligomycin that adds 3 μ m and the tubatoxin of 1 μ m, the variation of C2C12 cell OCR (OCR) is by Seahorse XF24 analysis-e/or determining, and represents C at the substrate row with %, ALCAT1 increases ROS level in the C2C12 cell, further aggravation when adding hydrogen peroxide.The C2C12 cell of overexpression ALCAT1 reduces in the face of oxidative stress plastosome copy number.E-H, the C2C12 cellular oxidation of overexpression ALCAT1 stress be increased by related gene expression, oxidation resistant expression of enzymes reduces, NADPH oxidase (Nox), dual oxidase, (Duox), glutathione peroxidases (Gpx), peroxiredoxin (Prdx), catalase (Cat), thioredoxin reductase (Txnrd), andsuperoxide dismutases (Sod).J, ALCAT1 mRNA expresses increase isolated myocardial cell in the time of in the face of oxidative stress.Separation is cultivated in the DMEM that the myocardial cell of C57/B16 mouse is being contained 10% foetal calf serum cultivates, added 100M oftert-butylhydroperoxide (T-Bu) or 0.5mM H202 or blank one hour, analyze the expression of ALCAT1 mRNA then with RT-PCR, beta-actin is as confidential reference items.Should be data from the measurement of three independent groups.* P＜0.05, * * P＜0.01 is with respect to control group.

Fig. 3 ALCAT1 disappearance mouse can avoid eating the fat and relevant insulin resistant of source property.Male ALCAT1-/-control mice of mouse and wild-type, the diet of continuous eight all feeding 60% fat, and measure weight increase, health is formed, glucose tolerance test and Regular Insulin carbohydrate tolerance test.*P＜0.05，**P＜0.01，n＝8-10。

Fig. 4 in C2C12 cell and mouse tissue ALCAT1 to the influence of signal path.A, the ALCAT1 overexpression is to the influence of Akt2 phosphorylation in the C2C12 cell.Add 0.1,1,10 and 100nM Regular Insulin 10 minutes respectively at ALCAT1 overexpression or the C2C12 cell that changes empty carrier over to, and analyze by Western blot and to change, analyze Akt (S473) phosphorylation and total AKT level with monoclonal antibody.B, separate ALCAT1-/-mouse and wild-type control group mice adipocyte add in the 10% foetal calf serum substratum at DMEM/F12 and cultivate, and with 0,1,10nM Regular Insulin was handled 10 minutes, then with immunoblotting detection Akt phosphorylation, and quantized.C-E analyzes liver with immunoblotting, and skeletal muscle and fatty tissue signal path are to the reaction of insulin stimulating.ALCAT1-/-mouse and wild-type mice overnight fasting, by abdominal injection Regular Insulin (1 unit/kg body weight) or PBS, and at back 10 minutes results tissues of injection.Liver, the protein lysate of fat and muscle is handled the back and is detected Akt with immunoblotting, AMPK, JNK, the phosphorylation of IRS1.Beta-actin is used as confidential reference items.

Fig. 5 improve ALCAT1-/-the mouse energy expenditure is with voracious, hypermetabolism, the activity of brown fat increases relevant.Male ALCAT1-/-diet of 60% fat of feeding in continuous 8 weeks of mouse and wild-type control group, and analyzed the intake of water and food.(A), body movement and energy expenditure (B), oxygen consumption rate (C), the form of respiratory exchange rate (D) white adipose tissue (WAT) and brown adipose tissue (BAT) is by H/E dyeing (E).*P＜0.05，**P＜0.01，n＝8-10。

Fig. 6 ALCAT1 disappearance has hindered liver cirrhosis and has strengthened the generation of liver fat acid oxidase.ALCAT1-/-mouse and the food rich in fat of feeding in continuous 24 weeks of wild-type control group, induce liver cirrhosis.Wild-type control group and ALCAT1-/-Mouse Liver section H/E staining analysis.With respect to the wild-type control group of liver cirrhosis (representing) with arrow, ALCAT1-/-group liver sample shows that big fat drips (being represented by arrow).From ALCAT1-/-mouse liver separates to be increased (C) with respect to the wild-type control group the plastosome obtain and reduces in the C12C12 of ALCAT1 stably express cell fatty acid oxidation ratio.**P＜0.05，**P＜0.01。

Fig. 7 ALCAT1 and diabetes B are to the active adjusting of mitochondrial ATP production rate and complicacy.A is from the COS-7 cellular segregation plastosome of transient expression ALCAT1 and measure the production rate of ATP.B, from stably express ALCAT1, empty carrier and shRNA disturb the C2C12 cellular segregation plastosome of ALCAT1 and measure the production rate of ATP.C, stably express ALCAT1, the C2C12 cell of empty carrier be with 0,1, and 10 and the Regular Insulin pre-treatment of 100nM 1 hour, separate mitochondria and measure ATP productivity then.E, from 50ug organize cracking measure the productive rate of ATP or F from 10 age in week the db/db diabetes mouse, the control of non-diabetic wild-type control group, and continuous 4 weeks of the mouse of db/db diabetes add Rother row ketone (rosiglitazone) (0.375mg/ days), and with of the explanation of Promega test kit according to manufacturers.G plastosome composite I activity is measured from stably express empty carrier or ALCAT1 gene C 2C12 cell.H from ALCAT1-/-extract plastosome and measure the composite I activity mouse or the wild-type control group mice liver.These data are from 3 cell samples or 5 independently experimentation on animals groups.* P＜0.05, * * P＜0.01 is compared with control group.

Embodiment

One, animal and reagent

Male and female C57BL/6 mouse, 4 to 6 weeks are available from Jackson Lab.All animals all live in one to be had round the clock the photoperiod, freely obtains water and standard mouse cage and research with in the high fat diet environment.Wherein 60% heat comes from lard or soya-bean oil.All animal vias that use in this research are crossed the approval of animal health mechanism, according to NIH agreement specified criteria.The use polyclonal antibody comprises antibody, phosphorylation AKT antibody (Ser473), phosphorylation AKT antibody (Thr308), AMPK Alpha antibodies, phosphorylation AMPK Alpha antibodies (Thr172), IRS-1 antibody, phosphorylation IRS1 antibody (Ser302) and phosphorylation stress activated protein kinase/JNK antibody (Thr183/Tyr185) of AKT, Cell Signaling Technology in this research.The polyclonal antibody of JNK1 is available from Santa Cruz biotech company, and the anti-rabbit igg horseradish peroxidase of donkey antibody is available from GEHealthcare.

Two, retrovirus generates stably express or the not enough C2C12 clone of ALCAT1

Mouse ALCAT1 total length encoding gene from plasmid pcDNA3.1 Flag-ALCAT1 subclone to entering p-BABEpuro (increase Bam HI and Sal I restriction enzyme site) retrovirus expression vector.Reorganization pBABEpuro-ALCAT1 and empty P-BABEpuro carrier are used to generate ALCAT1 overexpression and ONX blank recombinant retrovirus.Two mouse ALCAT1 siRNAs (siRNA), its a middle or short term the retrovirus clone, (5 ' TGCTGTTGACAGTGAGCGCGGACACAGTCCATTCTTTAATTAGTGAAGCCACAGAT GTAATTAAAGAATGGACTGTGTCCTTGCCTACTGCCTCGGA3 ') of hairpin RNA is at the pSM2C retroviral vector, available from Open Biosystems company, and be used for producing the short hairpin RNA interference mouse ALCAT1 expression that recombinant retrovirus is expressed.Recombinant retrovirus transfection when ONX cell 80% degrees of fusion wherein contains 122mM CaC ₁₂, 25mM HEPES, pH7.05; 5mM KCl; 6mM Dextrose; 140mM NaCl; 0.75mM Na ₂HPO ₄。Renewed bright DMEM substratum after the transfection in 10 hours.The retrovirus supernatant liquor is changeing 48 hours results then, and passes through one 0.2 micron membrane filtration.In order to screen stably express and ALCAT1 disappearance C2C12 clone, infected recombinant retrovirus C2C12 cell and screened with tetracycline after 48 hours.Clone with tetracycline resistance selects and enlarges.Immunoblotting and quantitative RT-PCR analysis have been verified at stable cell lines ALCAT1 expression level.

Three, immunoblotting assay

The ALCAT1 expression vector or the empty carrier of the stable transfection FLAG label of C2C12 cell, add 0,0.1,1.0,10 and the Regular Insulin of 100nM be respectively 10 minutes, and at RIPA cell pyrolysis liquid (20mM Hepes, 2mM EGTA, 50mM NaF, 100mM KCl, 0.2mM EDTA, 50mM β-glycerophosphate, 1.5mM Na 3VO4,10mM Na4VO7,1mMBenzamidine, 100ul Phosphatase Inhibitor Cocktail I, 1%Triton X-100,1.0mM PMSF), secondly centrifugal 20,000xg 5 minutes.This supernatant is with the total Akt of immunoblotting assay, phosphorylation Akt1/2, Erk1/2, and phosphorylation-ERK1/2.For the insulin signaling analysis of tissue samples, ALCAT1-/-mouse and control group mice overnight fasting, abdominal injection Regular Insulin (1 unit/kg body weight) or PBS then are after back 10 minutes euthanasia of injection.Handle tissue rapidly, freezing at liquid nitrogen.This tissue samples is pulverized at liquid nitrogen, in the homogeneity of RIPA damping fluid.Ice bath 30 minutes, 4 ℃ 10, centrifugal 10 minutes of 000xg, albumen is measured protein concentration with PIERCE BCA Protein Assay (Thermo).Equal protein matter (30ug) is used the SDS-polyacrylamide gel electrophoresis, transfers to pvdf membrane, the sealing of Tris-buffered saline 5% milk, anti-(1: 1000) 4 ℃ of refrigerator overnight.Two anti-(1: 2500) are at room temperature one hour then, develop with the ECLplus system at last.

Four, intracellular reactive oxygen and lipid peroxide are measured

The flow measurement that intracellular reactive oxygen generates at the C2C12 cell: use 2 ', 7 '-dichlorfluorescein-diacetate (dichlorofluorescein; Molecular probe) this material generates index as intracellular reactive oxygen.This material is assembled in cell, is produced 2 by the tenuigenin esterase hydrolyzed then, and 7-dichlorofluorescin is then with H ₂O ₂Reaction produces fluorescent substance 2,7-dichlorofluorescein (DCF).Cell is incubated in 37 ℃ of substratum that contain 5M DCFH-DA 30 minutes, cleans then, with 0.5 milliliter PBS suspension, and uses flow cytometry analysis.The influence 0.5mM H of reactive oxygen species in the oxidative stress pair cell ₂O ₂The DCF of 48 hours C2C12 cell of pre-treatment analyzes.In each measurement, minimum 20000 cells are used for analyzing.The tissue samples of lipid peroxidation is analyzed by the generation of measuring thiobarbituric acid reaction material (TBARS).Tissue sample is pulverized, and suspends at the RIPA lysate.TBARS output is by business-like kit measurement.Sample is analyzed in the 530-540 nanometer with microplate reader.

Five, real-time quantitative PCR analysis

Total cell or mouse tissue RNA obtain with TRIzol reagent.CDNA is synthetic with the Invitrogen SuperScript III first chain synthetic agent box.Oxidative stress and anti-oxidative defense reaction array and active oxygen real-time quantitative PCR are used in the real-time quantitative PCR analysis.Five house-keeping genes are used for each template concentrations internal parameter control group.Genetic expression relatively, the Ct=of calculation sample (Ct sample GENE) (Ct sample HKG).Then, genes involved table (RGE)=2power (Ct sample1Ct sample 2).The plastosome copy number is analyzed, and NDE is the Mitochondrial DNA mark, and cyclophilin is genome mark ND1: forward: 5 '-TGACCCATAGCCATAATATGATTT-3 ' is reverse: 5 '-CTCTACGTTAAACCCTGATACTAA-3 '; Cyclophilin: forward: 5 '-ACACGCCATAATGGCACTGG-3 ' is reverse: 5 '-CAGTCTTGGCAGTGCAGAT-3 ').

Six, intact cell oxygen consumption

Mouse sarcoplast (C2C12) crosses expresses ALCAT1 in the culture dish of the 100mm CO at 37 ℃ ₂Grow with the DMEM substratum of 1O%FCS and many anti-(5U/ml penicillin, 50 μ g/ml streptomycin, 3 μ g/ml puromycine) in the case.Use SOM-2PO2 Clark oxygen determination of electrode in the rate of consumption of intact cell oxygen.For each measurement, the results of the tryptic digestion of 3x106 C2C12 cell, and suspend again with 1.4ml DMEM substratum, add electrode then.Stir 1 minute balance, the base respiration effect is to weigh oxygen-consumption on average to surpass 5 minutes, is sequentially added into oligomycin (final concentration is 2.5g/ml), to trifluoro methoxycarbonyl cyanogen phenyl hydrazones (FCCP, final concentration is 2uM) enter electrode, breathe the highest breathing with measurement state 4.The rate of consumption of oxygen be expressed as natom oxygen/minute/1,000,000 cells.

Seven, isolate mitochondrial oxygen-consumption

Separation is placed on from the plastosome of liver and contains 250mM sucrose, 2mM KH2PO4, and 1mM EGTA is in and 20mMTrisHCl (pH 7.2) substratum.Hepatic mitochondria is prepared by standard differential centrifugation program.Mitochondrial protein assay test kit with Pierre Si and bovine serum albumin as standard.To the measurement of oxygen consumption, plastosome is incubated in (0.5-1mg/ml final concentration) cuvette and uses the Clark determination of electrode.All cut-and-try works are carried out in damping fluid below 37 ℃: 125mM KCl, 1mM EGTA, 2mM KH2PO4 and 20mM TrisHCl (pH 7.2).The breathing state of Kong Zhi non-phosphorylating (nonphosphorylating) is to carry out in different substrates (6.25mM glutamate/1.25mM malate or5mM succinate/0.5mM malate+1.25 μ M rotenone or 50 μ M palmitoyl-carnitine) in contrast.State 3 (phosphorylation breathing) adds 1mM ADP mensuration again.Oligomycin (1.25 μ g/mg albumen), a special mitochondrial ATP synthetase inhibitors is added to and measures the definite respiratory rate (state 4) of expense phosphorylation time-out in the mitochondrial suspension.The efficient of plastosome oxidative phosphorylation has been carried out assessment used state 3 to 4 than state, wherein (RCR RCR) under the situation without any kinetic control, carries out from upstream phosphorylation process the controlled oxidation phosphorylation.The bovine serum of no free fatty acids (FFA) is all used in all experiments, and having got rid of the free fatty acids in the bovine serum has energy inductive oxidative phosphorylation coupling.Mitochondrial respiratory titration FFA-BSA experiment has all been done in two groups of experiments.Minimum free-fat acid concentration albumin determines that best RCR is 3mg/ml (0.3%wt/vol, a data not shown).

Eight, mitochondrial ATP output capacity (MAPR)

The cell tryptic digestion washes twice at ice-cold PBS, and is suspended in 1 milliliter of homogenization buffer liquid (5mM MgCl2.6H2O, 1mM EDTA, PH 7.4 for 20mM HEPES, 140mMKCl), puts on ice then 3 minutes.4 ℃ of homogenate 600g centrifugal 5 minutes then, and supernatant is 10,000g, 4 ℃ centrifugal 10 minutes, the precipitation line plastochondria.To contain the damping fluid of mitochondrial particle suspension in homogeneity.The plastosome that is separated to is measured MAPR with ENLITEN ATP Assay system in 1420Multilabel Counter.Data are shown as nmol ATP/mg protein/min at last.

Nine, the XF bio-energy detects

Oxygen-consumption and glycolysis-amount are used the XF24 analysis-e/or determining.C2C12 cytotostatic transfection ALCAT1 expression vector or empty carrier are inoculated in the every hole of 24 porocytes and connect 20000 cell 250ul 10%FCS and antibiotic DMEM enlarging and hatched 20-24 hour at 37 ℃ then.Remove the DMEM growth medium after the experiment beginning and change 700ul detection substratum (low-buffer DMEM nutrient solution), and in advance 37 ℃ of heating.Made medium temperature and pH value reach balance in 30 minutes in the cultured cells incubator.Adding the mixing of measurement substratum at the XF24 analyser before measurement allowed oxygen partial pressure reach balance in 12 minutes.After the mixing, acidification rate (ECAR) outside oxygen consumption rate (OCR) and the extracellular is measured and was set up a benchmark value in 3 minutes.The substratum remix recovered normal oxygen tension and pH value in 2 minutes once more.70ul adds arrival ideal ultimate density in each hole after the base measurement.Next is to mix 2 minutes, to accelerate the egg of compound exposing cell.In general, following three the plain rates of each condition are measured respectively.Usually, detect cell at each and finally also made result standardization with the mensuration cell count with 0.25% tryptic digestion.

Ten, Fatty Acid Oxidation analysis

The Fatty Acid Oxidation rate is measured with hepatic mitochondria and C2C12 cell.Fatty Acid Oxidation is measured and has been used the bottle that seals at 0.4ml damping fluid (25mmol/l sucrose, 75mmol/l Tris-HCl, 10mmol/l KH2PO4,5mmol/l MgCl2, and1mmol/l EDTA, pH 7.4) supplemented with 5mmol/l ATP, 1mmol/l NAD+, 0.1mmol/l CoA, 0.5mmol/l L-carnitine, 0.5mmol/l L-malate and 25 μ mol/l cytochrome C.Blank is with ice-cold damping fluid.For plastosome (protein of 200ug), adding 0.1ml of 600 μ mol/l[14C] palmytoil-carnitine is at 37 ℃ the initial action after 5 minutes of hatching, and hatch and used 0.2ml of20%perchloric acid termination reaction in 15 minutes again for 37 ℃ then.For the measurement of Fatty Acid Oxidation in the intact cell, the C2C12 cell cultures is at the culturing bottle of 25 centimetres of diameters.When the cytogamy degree reaches 80-90%, these cells clean 3 times with cold PBS.Cell cultures is in containing 2%BSA low sugar DMEM.Add 0.1ml of 600 μ mol/l of[14C when beginning to react] palmytoil-carnitine.Then at 37 ℃ of 95%O ₂/ 5%CO ₂Incubation added 0.2ml20%perchloric acid termination reaction in 60 minutes again., add scintillation solution and measure [14] CO after 90 minutes in the room temperature vibrations ₂CO at room temperature after 90s, that [14] produce ₂The amount that generates.The Fatty Acid Oxidation rate be expressed as nmole/minute/gram albumen.

11, the activity measurement of citrate synthase

The Oxalacetic transacetase activity is used as the cyclophorase mark.Plastosome (200ug albumen) or cell homogenates (75ug total protein) add 100mM TrisHCl to, 1mM 5,5 '-dithiobis-2-nitrobenzoic acid, 3mM acetyl-CoA, pH 8, in the damping fluid of containing 0.2% (vol/vol) Triton X-100.Reaction originates in adding 1mMoxaloacetate, and initial reaction rate obtains by measuring the decline of 420nm absorbancy.The representation of activity of enzyme is U/ minute/milligram albumen.

12, plastosome composite I determination of activity

Plastosome and cell homogenates are measured plastosome composite I (the ubiquinone reductase enzyme of NADH) activity at the 50mM phosphate buffered saline buffer after pH 7.2 ultrasonications.Analytic liquid is 2mM of sodium cyanide, 2 μ g/ml antimycin A, the phosphate buffered saline buffer of 0.1 μ Mdecylubiquinone and 50mM phosphate buffer pH7.2.Plastosome sample (16 microgram) or cell sample (150 microgram total protein) are added in 1 milliliter of analytic liquid, and reaction is by the beginning of the NADH that adds 200uM.Reaction back is by measuring spectrophotometer minimizing assaying reaction speed to the absorbancy of NADH when 340nm.Active calculating is by calculating the disappearance coefficient of 6.22mM-1cm-1 NADH.Complex body I is active in the active stdn of citric acid, and expression mU/min/CS.

13, glucose tolerance test and insulin resistant test

Glucose tolerance test and insulin resistant test are what to carry out in the mouse that hunger is spent the night.After the fasting plasma glucose initial estimate, 2.5 gram glucose/kg body weight are irritated stomach.Abdominal injection 0.75 units of insulin/kg body weight.Measure blood sugar (instrument One Touch Ultra 2 blood sugar detection instrument) after 0,30,60,120 minutes taking glucose or Regular Insulin.

14, health is formed, energy expenditure, activity and food intake

Body fat and lean mass are measured in conscious mouse and are measured employing 1H-MRS with the fat and the lean mass of health.Every group of 7-8 mouse.With the intake of food/water of 3 days of metabolic cage measurement, energy expenditure, respiratory exchange ratio, body movement.Constant gas (0.6 liter/minute) is monitored by cage and with responsive under meter.Oxygen and concentration of carbon dioxide are monitored to calculate oxygen-consumption and RQ at the cage entrance and exit of sealing.Each cage was measured 1 minute every 15 minutes.Physical activity is measured with infrared equipment.

15, shotgun lipid diphosphatidylglycerol is analyzed

The method that lipid analysis is used as previously mentioned.In brief, carry out three mass spectroscopy (MS) by the Xcalibur system software analysis from C2C12 cell and animal tissues's TL.All mass spectrums and spectrum tandem mass spectrum are obtained automatically and are being operated under the Xcalibur sub-routine software under the self-defining sequence.Be scanned after the ion isolation of spectrum mass spectrum lipid.Every kind of corresponding quasi-molecular ions of molecule is by two-dimentional mass spectroscopy.The lipid of these integral parts is discerned by two tandem mass spectrum technology, monitors the output of neutral segmental loss or fragmention.Each module qualitative and quantitatively be by the air gun iipidomic.The diphosphatidylglycerol molecular species is determined by adding the double charge analysis.

16, statistical study

All data all are expressed as mean value ± SEM.Carrying out statistical is with the t check, with difference definite and wild-type and alcat knock out mice.

Claims (10)

1.ALCAT1 as the novel targets of drug treatment of diabetic, obesity and cardiovascular diseases and ALCAT1 biological characteristics as novel targets.

2. contain claim 1 described in the C2C12 cell ALCAT1 overexpression cause diphosphatidylglycerol disappearance similar to the pathologic process of metabolic trouble with diphosphatidylglycerol reconstruct.

3. contain overexpression ALCAT1 causes in the described C2C12 cell of claim 1 mitochondria dysfunction and oxidative stress.

4. containing the described ALCAT1 knock out mice of claim 1 makes up.

5. contain the described ALCAT1-of claim 1/-mouse is difficult for because diet induced and obesity (DIO) and insulin resistant.

6. contain the described ALCAT1 disappearance of claim 1 and improve insulin signaling.

7. contain claim 1 described ALCAT1-/-mouse strengthens energy expenditure, body movement and brown fat activity.

8. contain the described ALCAT1-of claim 1/-mouse shows and a kind ofly improves Fatty Acid Oxidation and may resist the liver cirrhosis of high-fat diet induced.

9. containing the described ALCAT1 of claim 1 lacks and improves mitochondrial function and suppress the activity of the productive rate of ATP and complicacy and insulin sensitizer seemingly.

10. contain the ALCAT1-of described increase linoleic acid content of claim 1 and anti peroxidation of lipid level/-mouse.

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