CN107412770A

CN107412770A - Strengthen the methods and applications of brown fat cell heat production efficiency

Info

Publication number: CN107412770A
Application number: CN201610349649.3A
Authority: CN
Inventors: 康建胜; 谢涛嵘
Original assignee: Shanghai Yu Jian Biotechnology Co Ltd
Current assignee: Shanghai Yu Jian Biotechnology Co Ltd
Priority date: 2016-05-24
Filing date: 2016-05-24
Publication date: 2017-12-01

Abstract

The invention provides the methods and applications of enhancing brown fat cell heat production efficiency, specifically, the invention provides a kind of purposes of heat production enhancing compound, for preparation of preparation or composition, the preparation or composition are used for the heat production efficiency for strengthening NE classes compound induction brown fat cell, wherein, the heat production enhancing compound is selected from the group：ATP classes compound, ATP synthetase inhibitors or its combination.The heat production enhancing compound of the present invention can significantly increase the heat production efficiency of norepinephrine class compound induction brown fat cell.

Description

Strengthen the methods and applications of brown fat cell heat production efficiency

Technical field

The present invention relates to biological technical field, in particular it relates to strengthen the side of brown fat cell heat production efficiency Method and application.

Background technology

For the homeothermal animal such as most of birds and mammal, tieed up in various complex environments Constant body temperature is held, it is necessary to efficient humidity control system.The constant balance for coming from heat production and radiating of body temperature State.Origin of heat in animal body mainly has four kinds of modes：Basic metabolism, body activity, cold stimulation draw The calorigenic action (including shivering thermogenesis and nonshivering thermogenesis) and the fuel factor of food risen.

Initial thermogenesis mechanism is to tremble during cold stimulation, is trembling to produce heat by musculature, but If it is exposed to for a long time in cold environment, then body temperature will be maintained by nonshivering thermogenesis.Brown fat Fat tissue (brown fat tissue, BAT) is the main heating source of nonshivering thermogenesis, particularly in new life In the animal of youngster and hibernation (Gesta et al., 2007).Brown adipose tissue, it is to be different from white adipose With a kind of cream-coloured fatty fatty existence form (Ussar et al., 2014), be distributed mainly under omoplate, Between omoplate, neck, chest, oxter, intercostal, around sustainer, around heart, groin and backbone The regions such as back and sympathetic ganglion top also have a small amount of distribution (Cannon and Nedergaard, 2004a).BAT is mainly made up of brown fat cell, and nucleus is centrally located, around wraps many fat drips, Mitochondria is in pelletiod, and number is various.There is abundant capillary and substantial amounts of sympathetic between brown fat cell Nerve endings fiber is distributed, abundant mitochondria and blood vessel make adipose tissue present brown ( 2009).In animal BAT total amount with distribution with species are different and the different developmental phases of animal and adaptation shape Condition shows different.

Thermogenesis in Brown Adipose Tissue is adjusted by central nervous system, the temperature that cold stimulation signal passes through body Receptor be delivered to brain cause corresponding sympathetic nerve release neurotransmitters (Nakamura and Morrison, 2008), most importantly norepinephrine (norepinephrine, NE).Norepinephrine acts on PKA signal paths in the adrenocepter activation downstream on brown fat cell, so as to activate mitochondria UCP1 (uncoupling protein 1) on inner membrance, the consumption of proton electrochemical potential energy is set to synthesize solution with ATP Coupling, energy are discharged in the form of heat.

When the energy of body intake exceedes consumption for a long time, unnecessary energy will be stored in the form of fat In body, obesity is caused in the course of time, and fat treatment is also concentrated mainly at 2 points：The intake of energy is reduced, Increase the consumption of energy.The research of slimming medicine before focuses on the intake reduced to energy, also there is some progress, But the medicine of such fat-reducing all lacks complete effect and Adverse event assessment, cause medicine application risk compared with Greatly.

Therefore this area is fat there is an urgent need to develop a kind for the treatment of that can increase energy expenditure and Small side effects Potential drug.

The content of the invention

It is an object of the invention to provide a kind of the latent for the treatment of obesity that can increase energy expenditure and Small side effects In medicine.

First aspect present invention provides a kind of purposes of heat production enhancing compound, for preparing a preparation or combination Thing, the preparation or composition are used for the heat production efficiency for strengthening NE classes compound induction brown fat cell, wherein, The heat production enhancing compound is selected from the group：ATP classes compound, mitochondrial complex V/ATP synthetase inhibitors, Or its combination.

In another preference, the ATP classes compound is selected from the group：ATP、ATP-γS、BzATP、α,β- Methylene adenosine triphosphate, 2- methylthioadenosines triphosphoric acid, ADP, UTP, UDP, MRS2690, UDP- grape Sugar, UDP- galactolipins or its combination.

In another preference, the mitochondrial complex V/ATP synthetase inhibitors are selected from the group：It is few mould Element, polygodial, orthovanadate or its combination.

In another preference, the preparation or composition also include NE class compounds.

In another preference, the NE classes compound is selected from the group：Norepinephrine, Cimaterol, Dobutamine, isoprel (isoprenaline), BRL37344, CL316243, GR265162X, L755507, SB251023, CGP12177 or its combination.

In another preference, the composition or preparation be additionally operable to prepare prevention and/or treatment it is fat or The medicine of obesity-related disease.

In another preference, the obesity-related disease is selected from the group：High fat of blood, type ii diabetes, fat Fat liver, hypertension, atherosclerosis, coronary heart disease, cholecystitis, OSAS, Children development caused by obesity is abnormal or it is combined.

Second aspect of the present invention provides a kind of method of the enhancing Into The Thermogenesis In Mammals of external non-therapeutic, bag Include step：

In the presence of NE classes compound and heat production enhancing compound, brown fat cell is cultivated, so as to strengthen The heat production of mammal, wherein, the heat production enhancing compound is selected from the group：ATP classes compound, mitochondria Compound V/ATP synthetase inhibitors or its combination.

In another preference, the mol ratio of the NE classes compound and heat production enhancing compound is 1-100：10-1000, it is preferred that 1-5：50-500, more preferably, 1-2：100-200.

In another preference, the activity of the NE classes compound is 0.01 μm of ol/L-10 μm of ol/L, It is preferred that 0.05 μm of ol/L-1 μm of ol/L, more preferably, 0.1 μm of ol/L-0.5 μm of ol/L.

In another preference, the activity of the heat production enhancing compound is 0.1-100 μ g/mL, compared with Goodly, 1-50 μ g/mL, more preferably, 5-20 μ g/mL.

In another preference, the mammal includes people.

In another preference, the mammal includes non-human mammal.

In another preference, the non-human mammal includes rodent, such as mouse, rat.

Third aspect present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition includes：

(i) NE classes compound；

(ii) heat production enhancing compound, the heat production enhancing compound are selected from the group：ATP classes compound, line grain Nanocrystal composition V/ATP synthetase inhibitors or its combination；With

(iii) pharmaceutically acceptable carrier.

In another preference, the component (i) and the mol ratio of component (ii) are 1-10：10-1000, compared with Goodly, 1-5：50-500, more preferably, 1-2：100-200.

In another preference, the component (i) and component (ii) account for described pharmaceutical composition gross weight 0.1-99.9wt%, it is preferred that 10-95wt%, more preferably, 50%-90wt%.

In another preference, described pharmaceutical composition also includes the compound of other enhancing Into The Thermogenesis In Mammals： Capsaicine, CL316243, dinitro benzene or its combination.

Fourth aspect present invention provides a kind of medicine box, and the medicine box includes：

(a) the first preparation containing NE class compounds；

(b) the second preparation containing heat production enhancing compound, the heat production enhancing compound are selected from the group：ATP Class compound, mitochondrial complex V/ATP synthetase inhibitors or its combination；With

(c) specification.

In another preference, (a) and (b) can place different vessels (or packaging) respectively or be placed in same appearance Device (or packaging).

In another preference, the formulation of the first described preparation and the second preparation is identical or different.

In another preference, the formulation of first preparation and the second preparation is independently selected from the following group：Capsule, Tablet, suppository, granule, oral liquid or intravenous injection (including lyophilized formulations).

In another preference, the concentration of NE class compounds is 0.01 μm of ol/L-10 in first preparation μm ol/L, it is preferred that 0.05 μm of ol/L-1 μm of ol/L, more preferably, 0.1 μm of ol/L-0.5 μm of ol/L；

In another preference, the concentration of NE class compounds is 0.01 in first preparation μm ol/1000g-10 μm of ol/1000g, it is preferred that 0.05 μm of ol/1000g-1 μm of ol/1000g, more preferably Ground, 0.1 μm of ol/1000g-0.5 μm of ol/1000g preparation.

In another preference, the concentration of heat production enhancing compound is 0.1-100 μ g/mL in the second preparation, It is preferred that 1-50 μ g/mL, more preferably, 5-20 μ g/mL；Or

In another preference, the concentration of heat production enhancing compound is 0.1-100 μ g/mL in the second preparation, It is preferred that 1-50 μ g/mL, more preferably, 5-20 μ g/mL.

Fifth aspect present invention provides a kind of method for the compound for screening enhancing brown fat cell heat production, bag Include step：

(a) a mitochondrial complex V/ATP synthetase inhibitors are provided as test compound；

(b) in test group, in cultivating system, in the presence of NE classes compound and the test compound, Brown fat cell T1 for a period of time is cultivated, detects the cultivating system toffee adipocyte of the test group Heat production degree Q1；

And in the absence of the test compound and other conditions identical control group, detect described in control group The heat production degree Q2 of cultivating system toffee adipocyte；

(c) heat production degree Q1 and heat production degree Q2 that previous step is detected are compared, so that it is determined that described Whether test compound is the compound for strengthening brown fat cell heat production；

Wherein, if heat production degree Q1 is significantly higher than heat production degree Q2, then it represents that the test compound is increasing The compound of strong brown fat cell heat production.

In another preference, the heat production degree of described detection brown fat cell includes what detection was selected from the group The change of one or more indexs：Mitochondrial membrane voltage change, the change of intracellular pH value or intracellular ATP it is dense Degree change.

In another preference, the heat production increase of described brown fat cell is shown as：Under mitochondrial membrane voltage Drop, intracellular pH value rise, and intracellular ATP concentration, which declines, to be reduced.

In another preference, " being significantly higher than " refers to heat production degree Q1/ heat production degree Q2 ratio >=1.5, Preferably >=2.0, more preferably >=2.5.

In another preference, described method is non-diagnostic and curative.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as implementation Example) in specifically describe each technical characteristic between can be combined with each other, so as to form new or preferable skill Art scheme.As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Fig. 1 shows FAD and NADH known absorption (dotted line) and fluorescence spectrum.

Fig. 2 shows being positioned in brown fat cell for temperature sensitive dye, rhodamine B methyl esters and rhodamine 800. Wherein, A:Rhodamine B methyl esters cellular localization (red)；B:The mitochondria positioning (green) of rhodamine 800； C:The DIC of cell；D:Two kinds of dyestuffs have good mitochondria common location in the cell, and small figure is local puts Big figure；E:The intracellular mitochondria Temperature Distribution that the ratio of fluorescence intensity is shown.

Fig. 3 shows the heat production of the brown fat cell of NE inductions.Wherein, A:Caused by 0.1 μM of NE The exothermic reaction of individual cells, 76% cell heat production, and the heat production amplitude of each cell is not quite similar (n=150 cell, 5 experiments)；B:Control cell (water for adding equivalent) exothermic reaction (n=97 cell, Test three times)；C:0.1 μM of NE can cause the exothermic reaction of primary brown fat cell, and integral fluorescence is strong The change of degree ratio only has less than 20%；D：The cell DIC figures of representative single experiment；E：0.1 μM NE handles precellular thermal imaging；F：The thermal imaging of cell when 0.1 μM of NE is handled 20 minutes, Part cell does not have obvious heat production to respond；G and H：Beta 3 receptor specific agonist CL316243 and β by Body wide spectrum activator Isoprenaline is only capable of the exothermic reaction for causing the primary brown fat cell in part.

Fig. 4 shows the expression of NE acceptors in brown fat cell.The primary cell of the 6th day is used Trizol methods extract RNA, reverse transcription cDNA, carry out the PCR of short-movie section with designed primer, 30 Circulation.Then product is entered into row agarose gel electrophoresis.By electrophoretogram it can be seen that the expression quantity of alpha-2 receptor It is extremely low.

Fig. 5 shows that SR 59230A suppress the exothermic reaction that NE triggers.Wherein, A:1 μM of SR59230A Broad spectrum inhibitors as NE acceptors can suppress the exothermic reaction (n=97 of cell caused by 0.1 μM of NE Individual cell, test three times)；B：0.1 μM of NE thorn under the conditions of existing for SR 59230A at 1 μM Sharp cell does not have exothermic reaction.

Fig. 6 shows that β1receptor plays a part of in heat production.Wherein, A:β1receptor inhibitor 70nM The exothermic reaction for the cell that 0.1 μM of NE triggers, (n=40 thin after CGP-20712 is pre-processed 30 minutes Born of the same parents)；B:, β1receptor suppress after, be capable of heat production Leukopenia (42%, n=180 cells, 5 experiments), and exothermic reaction can not continue, and " bell " curve is presented；C-E：NE before processings (C), The thermal imaging of cell when 20 minutes (D), 45 minutes (E), the cell of the part heat production temperature at the 45th minute Decline.

Fig. 7 shows that NE acceptor inhibitors reduce P-HSL (Ser563) phosphorylation.Wherein, A：1, negative pair According to；2,0.1 μM of NE are handled 30 minutes；3,1 μM of SR59230A are pre-processed 30 minutes, at NE Reason 30 minutes；4,70nM CGP-20712 are pre-processed 30 minutes, and 0.1 μM of NE is handled 30 minutes； B：P-HSL (Ser563) Relative phosphorylation degree (n=3, mean value ± SEM) under the conditions of inhibitor is existing.

Fig. 8 shows the heat production of the brown fat cell of high concentration NE inductions.Wherein, A-B:HSL-563 Phosphorylation with concentration increase and increase, (n=3, mean value ± SEM)；C：It is single caused by 10 μM of NE The exothermic reaction (n=110 cell, 5 experiments) of individual cell, 81% cell is heat production；D：10μM NE triggers the slightly strong cell exothermic reactions of than 0.1 μM NE.

Fig. 9 shows the UCP1 positioning and expression of brown fat cell.Wherein, A:Brown fat cell UCP1 Immunofluorescence dyeing；B:Brown fat cell mitochondrial protein cromoci immunofluorescence dyeing；C:UCP1 With cromoci binary channels overlay chart, it is seen that UCP1 has very strong common location with cromoci；D： UCP1 and cromoci common location partial enlarged drawing, it is seen that clearly bar-shaped and chondriosphere；E： Cell UCP1 immunofluorescence dyeing figures under 40x times of mirror, all cells have UCP1 expression；F：With E Cromoci immunofluorescence dyeing figure corresponding to figure；H：UCP1 and cromoci bilateral trace overlap Figure；H：The DIC figures of cell.

Figure 10 shows ATP and the enhancing brown fat cell heat production of NE collective effects.Wherein, A：0.1μM The exothermic reaction for the individual cells that NE and 10 μM of ATP collective effect triggers, 95% cell is heat production (n=158 cell, five experiments)；B：Compared with the heat production that NE triggers, ATP and NE collective effects Make the overall heat production of cell substantially increase, and ATP then only has fainter reaction in itself；C：ATP with NE collective effects make the average heat production amplitude of heat production cell dramatically increase (mean value ± SEM)；D and E： ATP- γ S make the average heat production of heat production cell dramatically increase (mean value ± SEM) with NE collective effects.

Figure 11 shows that 0.1uM NE trigger the rise of brown fat cell Calcium ion, the calcium in cell cytosol Measured with dyestuff fura-AM, cell is put into 5 μM of Fura-AM tyrode solution, 37 DEG C of trainings Support and dyed 30 minutes in case, cell takes out to be placed in tyrode solution and observed.Wherein, A：0.1μ The Calcium ion reaction (n=54 cell, testing three times) of individual cells caused by M NE；B：Normalized list The calcium change of individual cell, has a rising drastically, then declines and maintain certain level；C：Institute The overall calcium for having cell changes；D：Fura2-AM 340nm excite fluorescence；E：Fura2-AM 380nm Excite fluorescence；F-G：Two passage overlay charts, 0.1 μM of NE before processing (D), processing 15 minutes when (F).

Figure 12 shows that NE causes the calcium of intracellular mitochondrial to increase.Primary brown fat cell is separated Carry out electricity afterwards to turn, the 3-7 days Germicidal efficacies.Wherein, A：PcDNA-4mtD3cpv cpVenus173 are glimmering Light；B：During NE processing, the calcium change of the mitochondria of individual cells；C：The line of all cells during NE processing Plastochondria calcium change (n=16 cell, four experiments).

Figure 13 shows that NE triggers the release of endocytoplasmic reticulum calcium.Wherein, A：pcNDA-D1ER Citrine Fluorescence；B：During NE processing, the calcium change of the endoplasmic reticulum of individual cells；C：All cells during NE processing Endoplasmic reticulum calcium change (n=18 cell, four experiments).

Figure 14 shows that brown fat cell heat production consumes intracellular reduction potential.Wherein, A：440nm swashs FAD fluorescence caused by hair；B：340nm excites common fluorescence signal caused by FAD and NADH simultaneously；C： The precellular redox state of NE processing；D：The redox state of cell when 15 minutes after NE processing； E：The change (n=60 cell, four experiments) of the redox state of cell when NE is stimulated；F：Single Test the redox state (n=20 cell) of individual cells；G：Normalized data show that cell is overall Redox state change.

Figure 15 shows that NE processing triggers intracellular ATP change.Wherein, A：AT1.03YFP fluorescence； B：During NE processing, the endochylema ATP changes of individual cells, when NE is added, intracellular ATP declines suddenly； C：The endochylema ATP changes (n=29 cell, four experiments) of all cells during NE processing.

Figure 16 shows that NE and ATP triggers intracellular mitochondrial ATP change.Wherein, A：mt-AT1.03 YFP fluorescence；B：During NE processing, ATP changes in the mitochondria of individual cells, when NE is added, mitochondria Interior ATP changes have larger fluctuation；C：Mitochondrial ATP change (the n=26 of all cells during NE processing Individual cell, four experiments).

The change and heat production of mitochondrial membrane voltage, intracellular pH value when Figure 17 shows 0.1 μM of NE processing Relation.Wherein, A：During 0.1 μM of NE processing cell, the change of the membrane voltage of cell, 55.6% cell Mitochondrial membrane voltage declines (n=150 cell, five experiments)；B：Mitochondrial membrane voltage change and cell are most The scatter diagram of whole heat production state, the cell that mitochondrial membrane voltage declines all is heat production, and mitochondrial membrane voltage It is heat production that the cell of rising, which then only has part cell,；Heat production and the change of membrane voltage are in obvious negative correlation (r =-0.73)；C：During 0.1 μM of NE processing cell, there is (solid dot) in advance or do not having (hollow dots) Add under the conditions of 10 μ g/mL oligomycin, the scatterplot of the change and the change of intracellular pH value of the membrane voltage of cell Figure.

Figure 18 shows the brown fat cell heat production that rotenone induced NE and the influence of membrane voltage.Wherein, A：5 μM of rotenone are added in 30 minutes in advance tyrode solution, after adding 0.1 μM of NE, carefully Born of the same parents' exothermic reaction is obvious, and all heat production of almost all of cell, but exothermic reaction does not continue；B:Carefully The W-response (n=22) of born of the same parents；C：The change of the membrane voltage of cell.

Figure 19 shows the brown fat cell heat production that oligomycin induced NE and the influence of membrane voltage.Wherein, A：10 μ g/mL oligomycin are added in 30 minutes in advance tyrode solution, after adding 0.1 μM of NE, Cell exothermic reaction is obvious, and all heat production of all cells；B:The W-response (n=25) of cell；C： The change of the membrane voltage of cell, the membrane voltage of all cells have reduction.

Figure 20 shows that ATP is probed into NE humidification mechanism.Wherein, A：0.1 μM of NE and 10 μ M ATP processing brown fat cell 30 minutes；B：NE and cell during ATP Co stituations redox The change of state when NE is individually stimulated compared with (four times experiment)；C：NE and NE+ATP mitochondrial membrane voltages Decline the ratio with heat production.

Embodiment

The present inventor unexpectedly develops a kind of enhancing noradrenaline first by in-depth study extensively A kind of heat production enhancing chemical combination of the heat production efficiency of chlorins compound (NE classes compound) induction brown fat cell Thing.Specifically, heat production enhancing compound includes ATP classes compound and/or ATP synthetase inhibitors, this It is brown that the heat production enhancing compound of invention can significantly increase norepinephrine class compound (NE classes compound) induction The heat production efficiency of color adipocyte.On this basis, the present inventor completes the present invention.

Term

As used herein, term " mitochondrial complex V/ATP synzyme ", " mitochondrial complex V ", " ATP Synzyme " is used interchangeably, and refers to the enzyme that ATP is synthesized on mitochondrial inner membrane.

As used herein, " MRS2690 " refers to pyrophosphoric acid 1- α-D- glucopyranose base ester 2- [(4'- first Base is thio) uridine -5 "-yl] ester disodium salt (Diphosphoric acid 1- α-D-glucopyranosyl ester 2-[(4'-methylthio)uridin-5”-yl]ester disodium salt)。

As used herein, " BRL37344 " refers to (R*, R*)-(±) -4- [2- [(2- (3- chlorphenyls) - 2- ethoxys) amino] propyl group] phenoxy acetic acid ((R*,R*)-(±)-4-[2-[(2-(3-Chlorophenyl)-2-hydroxyethyl)amino]propyl ]phenoxyacetic acid)。

As used herein, " CL316243 " refers to 5- [(2R) -2- [[(2R) -2- (3- chlorphenyls) -2- Ethoxy] amino] propyl group] -1,3- benzodioxole -2,2- dicarboxylic acids (5-[(2R)-2-[[(2R)-2-(3-Chlorophenyl)-2-hydroxyethyl]amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylic acid)。

As used herein, " GR265162X " refers to document (Mouse beta 3a-and beta 3b-adrenoceptors expressed in Chinese hamster ovary cells display Identical pharmacology but utilize distinct signalling pathways, Br J Pharmacol.2002Apr；135(8):1903-14.) in the compound mentioned.

As used herein, " L755507 " refers to 4- [[(hexylamine) carbonyl] amino]-N- [4- [2- [[(2S) -2- Hydroxyl -3- (4- hydroxyphenoxies) propyl group] amino] ethyl] phenyl]-benzene sulfonamido (4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4-hyd roxyphenoxy)propyl]amino]ethyl]phenyl]-benzenesulfonamide)。

As used herein, " SB251023 " refers to 4- [4- [2 (S)-hydroxyl -3- [3- (4- hydroxyphenoxies) Propylcarbamic] cyclopentyl-methyl] phenoxymethyl] (phenyl) phosphonic acids (4-[4-[2(S)-Hydroxy-3-[3-(4-hydroxyphenoxy)propylamino]cyclopentyl methyl]phenoxymethyl](phenyl)phosphonic acid)。

As used herein, " CGP12177 " refer to 4- [3- [and (1,1- dimethyl ethyl) amino) -2- hydroxyls Propoxyl group] -1,3- dihydro -2H- 2-ketone benzimidaozole hydrochlorides (4-[3-[(1,1-Dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-b enzimidazol-2-one hydrochloride)。

Heat production strengthens compound

As used herein, term " heat production enhancing compound " refers to ATP classes compound, the suppression of ATP synzyme Preparation or its combination.In addition, described heat production enhancing compound can also be with NE classes compound and containing Pharmaceutically acceptable carrier is combined into the pharmaceutical composition with enhancing brown fat cell Thermogenic Activity.

It is not particularly limited suitable for the ATP classes compound of the present invention, representational example is included (but not It is limited to)：ATP, ATP- γ S, BzATP, α, β-methylene adenosine triphosphate, 2- methylthioadenosines triphosphoric acid, ADP, UTP, UDP, MRS2690, UDPG, UDP- galactolipins or its combination.

Suitable for the present invention the ATP synthetase inhibitors be not particularly limited, representational example include (but It is not limited to)：Oligomycin, polygodial, orthovanadate or its combination.

It is not particularly limited suitable for the NE classes compound of the present invention, representational example is included (but not It is limited to)：Norepinephrine, Cimaterol, dobutamine, isoprel (isoprenaline), BRL37344, CL316243, GR265162X, L755507, SB251023, CGP12177 or its group Close.

When the heat production enhancing compound of the present invention is applied in combination with NE analogs, heat production enhancing compound and NE Ratio between analog does not have any restrictions.Generally, each component should meet its minimum valid density. In a preference, the heat production enhancing compound and the minimum effective concentration of NE analogs are as follows：

Typically, in the present invention, the minimum effective concentration of the heat production enhancing compound is 0.1-100 μ G/mL, it is preferred that 1-50 μ g/mL, more preferably, 5-20 μ g/mL.

Typically, in the present invention, the minimum effective concentration of the NE classes compound is 0.01 μm of ol/L-10 μm ol/L, it is preferred that 0.05 μm of ol/L-1 μm of ol/L, more preferably, 0.1 μm of ol/L-0.5 μm of ol/L.

Generally, the heat production enhancing compound and the mol ratio of NE analogs are 1-100：10-1000, compared with Goodly, 1-5：50-500, more preferably, 1-2：100-200.

Heat production enhancing compound of the present invention can significantly increase NE classes compound induction brown fat cell Heat production efficiency, it can generally improve about 50% or higher so that the total yield thermal efficiency averagely reaches about 75% or higher (such as 90%).

The method of the compound of screening enhancing brown fat cell heat production

Present invention also offers a kind of method for the compound for screening enhancing brown fat cell heat production, including step：

(a) an ATP synthetase inhibitors are provided as test compound；

(c) Q1 and Q2 detected previous step is compared, so that it is determined that the test compound whether It is the compound for strengthening brown fat cell heat production；

Wherein, if heat production degree Q1 is significantly higher than heat production degree Q2, then it represents that the test compound is Strengthen the compound of brown fat cell heat production.

In a preferred embodiment, described method also includes step (d)：By identified increasing in step (c) The compound of strong brown fat cell heat production is applied to animal, and determines its influence to animal heat production.

In a preferred embodiment, methods described also includes determining the one of heat production degree with temperature sensitive dye image method (concentration of such as mitochondrial membrane voltage change, the change of intracellular pH value, and/or intracellular ATP becomes individual or multiple indexs Change).

In another preference, the dyestuff used in the temperature sensitive dye image includes rhodamine 800 and rhodamine B The combination of methyl esters.

Preparation and pharmaceutical composition

Present invention also offers a kind of preparation or composition, and they include described heat production enhancing compound and NE Class compound and optional other carriers or excipient.

Preferably, described composition is pharmaceutical composition, food compositions, Halth-care composition etc..

By taking pharmaceutical composition as an example, pharmaceutical composition of the invention is including pharmaceutically acceptable carrier and effectively Measure two active components：(i) heat production enhancing compound and (ii) NE class compounds described in.

As used herein, term " effective dose " or " effective dose " refer to that people and/or animal can be produced Function or amount that is active and being received by people and/or animal.

As used herein, the composition of " pharmaceutically acceptable carrier " applies to people and/or mammal And without excessive bad side reaction (such as toxicity, stimulation and allergy), i.e., with rational benefit/wind The material of dangerous ratio.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including each Kind excipient and diluent.

The pharmaceutical composition of the present invention contains the active component of the invention of safe and effective amount and pharmaceutically acceptable Carrier.This kind of carrier includes (but being not limited to)：Salt solution, buffer solution, glucose, water, glycerine, second Alcohol, and combinations thereof.Usual pharmaceutical preparation should match with administering mode, the agent of pharmaceutical composition of the invention Type is injection, oral formulations (tablet, capsule, oral liquid), transdermal agent, sustained release agent.Such as use physiology salt Water or the aqueous solution containing glucose and other assistant agents are prepared by conventional method.Described drug regimen Thing preferably aseptically manufactures.

The effective dose of active component of the present invention can be with the pattern of administration and the serious journey of disease to be treated Degree etc. and change.The selection of preferable effective dose can by those of ordinary skill in the art according to various factors come It is determined that (such as passing through clinical test).Described factor includes but is not limited to：The medicine generation of described active component Kinetic parameter is such as bioavailability, metabolism, half-life period；The serious journey of the disease to be treated of patient Degree, the body weight of patient, the immune state of patient, the approach etc. of administration.Generally, when the present invention activity into Divide daily with about 0.00001mg-100mg/kg the weight of animals (preferably 0.0001mg-10mg/kg animal bodies Weight, more preferably 0.001mg-1mg/kg the weight of animals) dosage give, gratifying effect can be obtained. For example, by an urgent demand for the treatment of situation, dosage separated several times can be given daily, or by dosage press than Reduce on example ground.

Pharmaceutically acceptable carrier of the present invention includes but is not limited to：Water, salt solution, liposome, fat Matter, albumen, Protein-antibody conjugate, peptide matters, cellulose, nanogel or its combination.Carry The selection of body should match with administering mode, and these are all known to one of ordinary skill in the art.

Present invention also offers the purposes of described pharmaceutical composition, the heat production for (i) enhancing brown fat cell Efficiency；(ii) prevent and/or treat the relevant diseases such as obesity.

Main advantages of the present invention include：

(1) present invention firstly discovers that heat production strengthens compound (such as ATP classes compound, and/or ATP synzyme Inhibitor) can significantly increase NE classes compound induction brown fat cell heat production efficiency, heat production efficiency energy Enough reach 75% or higher.

(2) present invention firstly discovers that heat production can be strengthened into compound, (such as ATP classes compound, and/or ATP are closed Into enzyme inhibitor) prevent with NE class chemical combination Internet of Things and/or treat the relevant diseases such as obesity.

(3) present invention firstly provides mitochondrial membrane voltage, intracellular pH value, intracellular ATP concentration, cell are temperature sensitive The change of the indexs such as the cell temperature that dyestuff is shown can be used for screening and carry out obesity using brown fat heat production The medicine of disease treatment.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to manufactory Condition proposed by business.Unless otherwise indicated, otherwise percentage and number are percentage by weight and parts by weight.

Universal method and associated materials

1. brown fat cell original cuiture

Material therefor and reagent

3-4 weeks mouse (C57, male, western pul-Bi Kai), long filament dropper (two kinds of thickness), culture medium (DMEM), Hyclone (FBS), calf serum (NCS), PBS (pH7.4) and penicillin streptomycin (Pen Strep) All it is available from Thermo Fisher companies of the U.S., clostridiopetidase A II and cytarabin (Ara-C) purchase From Sigma-Aldrich, Matrigel is purchased from BD Biosciences companies of the U.S., glass Piece Coverslips is purchased from Thermo Fisher companies of the U.S..

Specific test method

1) by 4 DEG C of jellies of mouse overnight, it can't help drinking water.General three mouse are cooked 8 dish；

2) two 6cm dish are taken to be separately added into the 10mL of 37 DEG C of preheatings PBS and 4%NCS DMEM；

3) by mouse break neck put to death, ethanol for disinfection soak 10min, during this period by surgical scissors and tweezers Sterilized, be put in sterilized water with ethanol for disinfection；

4) mouse back is cut off to the brown adipose tissue taken out at shoulder blade, is put in PBS；

5) white adipose tissue is removed in PBS, brown adipose tissue is put in 4%NCS DMEM；

6) tissue is drawn onto in EP pipes, shredded with scissors, general 1mm or so fritter；

7) it is transferred again into 15mL centrifuge tubes, 10mL 4%NCS DMEM are washed once, sop up supernatant；

8) the clostridiopetidase A II (in PBS) of 1mL 1%, (clostridiopetidase A II use is added in 4mL 4%NCS DMEM PBS pH7.4 are prepared, packing, -20 DEG C of preservations, avoid freezing repeatedly), filtering, add in tissue；

9) 37 DEG C, 30min, small water-bath is placed on wiggly shaking table, speed 10, herein Period, long filament dropper mouth is burnt with alcolhol burner mellow and full, thin mouth burns general 0.5mm or so；

10) digestive juice is sopped up, 4%NCS DMEM are washed once；

11) 2mL 4%NCS DMEM, blown and beaten 5 times or so with thick mouth dropper, supernatant 1 is sopped up, weight New to add separating liquid, thick mouth dropper is blown and beaten 5-7 times, then is blown and beaten general 7 times or so with thin mouth dropper；

12) secondary supernatant is sucked into new centrifuge tube, remaining precipitation can be with if more Blown and beaten three times, to obtain as far as possible more cells；

13) 900g, 9min are centrifuged, obtains sedimentation cell；

14) precipitation is resuspended with 5%FBS DMEM, be taped against on cover glass；

15) 5%FBS DMEM 2mL are added after 2-3 hours；

16) liquid is changed within second day, partly changing liquid with the 5%FBS DMEM containing 4 μM of ARA-C (it is thin to suppress precursor The growth of born of the same parents)；

17) liquid is partly changed after once with 5%FBS DMEM within every 2 days, can when typically changing liquid for the second time Impurity is removed so that cell surface is gently blown and beaten, it can be seen that the adipocyte of the differentiation of separation is firmly Patch on the cover slip, cell now, which can be brought, tests, and the cell of general 3-8 days, which does experiment, is Out of question, the time is grown again, and it is big that fat drips will merge change.

2. brown fat cell transfects

Experiment material and instrument

1) plasmid of high quality：260:280>1.8, concentration is more than 1mg/mL (plasmid used and source are shown in Table 1)；

2) electroporation：BTX companies ECM830 square wave electricity transfers from one department to another to unite；

3) electricity turns liquid：20mM Hepes,135mM KCl,2mM MgCl₂, 0.5%Ficol 400,1%DMSO, pH 7.6；

4)20mM ATP:50mM Glutathione storing liquids (pH~7.4) .100x；

5) 0.4cm electricity revolving cup.

Electricity is gone to step

Electricity transfers to be put to room temperature with all reagents and all sterilized processing of material, required solution.

1) the mouse primary adipocyte separated washed once with PBS, 900g is centrifuged 9 minutes, and electricity turns Cell density need to reach 1~1.5x10⁶Individual cells/ml；

2) 100x ATP and Glutathione storing liquids are added into electricity to turn in liquid；

3) 10-40 μ g plasmids are added to electricity to turn in liquid；

4) turn liquid with about 420 μ l electricity and cell is resuspended；

5) electricity containing plasmid and cell is turned into liquid to be added in 0.4cm electricity revolving cups, mixes, close the lid；

6) shocked by electricity rapidly, condition 10ms, 115V square wave, 2 subpulses, time interval 1s；

7) 2mL 10%NCS DMEM are added, are mixed, 900g is centrifuged 9 minutes；

8) it is resuspended with normal incubation medium 600ul, 100 μ l/ cover glasses；

9) each culture dish (3.5cm) adds 2mL culture mediums, incubator culture 24 hours, partly changes liquid, adds Enter ARA-C to 2 μM of final concentration.

The plasmid of embodiment is conventional plasmid, and source is shown in Table 1.

Table 1

3. immunofluorescence dyeing

Immunofluorescence dyeing experiment material and instrument

Vavuum pump (Cat No are used in experiment：) and shaking table (Cat No gi-802：Ts-8) it is purchased from its woods shellfish of Jiangsu Haimen That instrument manufacturing Co., Ltd.

Solution used in immunofluorescence dyeing and reagent are as follows：

1) fixer FSB：Formaldehyde, 10%, methanol free, Ultra Pure are purchased from Polyscience Inc(Cat No：04018), other reagents are purchased from Sigma companies；

2) prepared with 1XPBS and contain 4%formaldehyde, 4%sucrose FSB；

3) confining liquid：0.5% Triton X-100 and 5% lowlenthal serum is added in 1X PBS；

4) mountant：Fluoromount-G is purchased from Southern Biotech companies (Cat No：0100-01)；

5) primary antibody：Anti-UCP1 (#U6382, Sigma, more anti-), anti-Phospho-HSL (Ser563, #4139, CST is more anti-), anti-cromocis (#556432, BD Pharmingen monoclonal antibodies)；

6) fluorescence secondary antibody：Purchased from Thermo Fisher companies.

Immunofluorescence dyeing experimental method

Immunofluorescence dyeing experimental procedure is as follows：

1) culture medium in net culture dish is blotted with vavuum pump, is washed three times with PBS；

2) 1mL fixer FSB is added in each culture dish, room temperature fixes 30 minutes on shaking table, Ran Houyong PBS is washed three times；

3) 1mL confining liquids are added in each culture dish, room temperature is closed 30 minutes on shaking table；

4) 1 is pressed with confining liquid:400 dilution proportion primary antibody, 4 DEG C are rocked overnight on shaking table；

5) washed three times with PBS, dilute fluorescence secondary antibody with containing 2% serum and 0.5%Triton X-100 PBS, Dilution ratio is 1:400；

6) mountant is dripped in slide center, cover glass is picked up with tweezers, exhausted residual night with blotting paper, gently Light back-off not stay bubble, be sealed cover glass edge with transparent nail polish on slide.

4. dyestuff loads and cell imaging

Solution used in living cells IMAQ is Tyrode：NaCl, 8.4738g, KCl, 3mL (1mol/L), HEPES, 2.383g, glucose, 1.8g, it is dissolved in 1L ddH₂In O, pH7.4.Agents useful for same is purchased From Sigma companies.Rhodamine B (RhB), rhodamine 800 (Rh800) and CCCP are purchased from the U.S. Sigma-Aldrich companies, norepinephrine (NE) are purchased from Santa Cruz companies of the U.S., SR-59230A is purchased from Abcam companies of Britain, and CGP-20712 is public purchased from Britain Tocris Bioscience Department.

Cell is in the tyrode solution of the rhodamine B methyl esters containing 30nM and rhodamine 800,33 DEG C Contaminate 1 hour altogether.High-definition picture is then contaminated altogether with 50nM dyestuffs.The puppet that rhodamine B methyl esters passage is set For coloured silk for red (559nm is excited, and 575-620nm receives light), the puppet that the passage of rhodamine 800 assigns is color to be green Color (635nm is excited, 655-755nm receive light), for shooting 40 ×/0.95 object lens 512 of Time Continuous image × 512 point resolutions, the acquisition of high-definition picture is then with 100 ×/1.4O object lens 1600 × 1600 point minute Resolution (view data is 12 bits).

5. intracellular pH is imaged

Intracellular pH imagings use 5 μM of 37 DEG C of SNARF-1AM (being purchased from Thermo Fisher companies of the U.S.) 30min is contaminated, tyrode solution is washed once, is placed in 2mL tyrode solution, 33 DEG C of constant temperature.559nm Excite, it is pH relative values that 655-755nm, which receives light with the 575-620nm ratios for receiving light,.Time Continuous image The for shooting 40 ×/point resolution of 0.95 object lens 512 × 512 (view data is 12 bits).

6. redox ratio value and calcium imaging

Oxidation-reduction potential uses with Calcium ion imaging and just puts microscope, purchased from Olympus.Fura2-AM is purchased from Thermo Fisher companies of the U.S..

Calcium imaging is washed once, is placed in using 5 μM of Fura2-AM, 37 DEG C of dye 30min, tyrode solution In 3.5cm culture dishes, 4mL tyrode solution, 33 DEG C of constant temperature.Water bath with thermostatic control and medicine system are Self-control.

The measurement of oxidation-reduction potential is then to have different excite and transmitted wave according to intracellular FAD and NADH It is long：Exciting for NADH excites at 350nm under normal circumstances, and the maximum of emission spectrum is in 450nm To between 470nm.FAD exciting light is 450nm, and the maximum of emission spectrum is in 520nm or so (figures 1).During experiment, due to being excited at 340nm, it is limited that 450nm to 470nm receives light, it is impossible to enters well The processing of row follow-up data, therefore expand the scope for receiving light, the FAD's that part is excited at 340nm Fluorescence also have received together.Reflect endocellular metabolism state using FAD/ (FAD+NADH) fluorescence ratio Change.(view data is for for shooting the 40 of the Time Continuous image ×/point resolution of 0.8 hydroscope 512 × 512 16 bits).

The cell imaging instrument of table 2 and software and effect

Parameter used in the cell imaging of table 3

7. Data Collection and processing

Cell is held in 33 DEG C of constant temperature during imaging, and the time interval of imaging is 30s, adds at the 11st NE processing, inhibitor then add for 20 minutes before taking pictures.

After background correction, it would be desirable to handle two passages (thermal imaging Rh800/RhB-ME, calcium imaging 340/380th, oxidation-reduction potential FAD/ (FAD+DANH), fret CFP/CFP) numerical value do it is point-to-point do ratio, In order to reduce noise, point of the ratio less than 1.5 of signal value and level of noise removes, remaining to carry out 5 × 5 It is average.According to 3-sigma rules, the extremum (99.7% tolerance interval) of ratio is removed.Each cell Ratio be the average value for having on this cell a ratio.The stable shape of cell is represented with the numerical value of before processing State does the normalized of numerical value, then the response value Δ r of cell can use formula Δ r=(r_t-r₀)/r₀ To calculate, r in this formula_tIt is to react the average value of last five minutes, r₀It is the flat of the initial state before dosing Average.

Data used in the present invention are average value ± s.d, and average value ± s.e.m is then used in figure.

8. Western blotting

Western blotting (western blot) experiment material and instrument

Triton X-100, NaTDC (DOC), albumen and inhibitors of phosphatases Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 2 and Phosphatase Inhibitor Cocktail 3 is available from Sigma companies, and protein concentration detection kit is determined purchased from health for century BCA albumen Measure kit (Cat No：CW0014), 12% pre-prepared colloid is purchased from Wen Yuange bio tech ltd (Cat No： P05), page gel electrophoresises loading is that the SDS-PAGE Loading Buffer that health is century (are gone back Original, 5x) (Cat No：CW0027A), albumen marker is the PageRuler Prest of fermentas companies Protein Ladder(Cat No：26616), Western blot are purchased from PE companies (Cat with 0.2 μm of NC film No：Nba083g001ea), exposure is X-OMAT BT X-rays (Cat No with X-ray：XBT-1), nitrite ion For Pierce ECL Western Blotting Substrate (Cat No：32109), Tissue Culture Dish is purchased from Nunc companies, page gel-electrophoretic apparatuses are purchased from Bio-Rad companies, and the miniature vertical electrophoresis tanks of VE 180 are purchased from day energy Company, transferring film instrument are the XCell II of Thermo Fisher companies^TMBlot Module(Cat No：EI9051), Refrigerated centrifuge is Heal Force companies Neofuge 23R.

Use antibody：

Primary antibody：Anti-Phospho-HSL (Ser563, #4139, how anti-) and anti-HSL (#4107, how anti-) Purchased from Cell Signaling Technology (CST) company.Anti-actin (#264267, abmart, it is single It is anti-).

Secondary antibody：Rabbit secondary antibody is purchased from GE companies (#NA934-1ML), and mouse secondary antibody is purchased from CST companies (#7076S).

Solution used in Western blot experiments is as follows, and wherein agents useful for same is purchased from sigma companies.

1)1XPBS：KH₂PO₄0.24g, Na₂HPO₄1.44g, NaCl 8.0g, KCl 0.2g, pH to 7.4, ddH₂O is settled to 1L；

2)10X TBS：NaCl 80g, KCl 2g, Tris 30g, pH 7.4, ddH₂O is settled to 1L；

3) 10X transferring films liquid：Tris 29.027g, glycine 144.1344g, ddH₂O is settled to 800mL, Used time adds 200mL methanol per 1L；

4) 10X electrophoresis buffer：Tris 30g, glycine 144g, SDS 10g, pH 8.4, ddH2O It is settled to 1L；

5)TBST：Add 500 μ l Tween20 per 1L TBS；

6)100X PMSF：1.74g PMSF are dissolved in 100mL isopropanols, dispense and preserve after dissolving；

7) lysate：X-100 containing 1%Triton and 1%DOC 1XTBS；

8) 10% ammonium persulfate：0.1g ammonium persulfates are dissolved in 1mL ddH₂In O.

Western blotting (western blot) experimental method

Western blotting (western blot) experimental procedure is as follows：

1) brown fat cell of 3-7 days is subjected to agent-feeding treatment.The general pre-add of inhibitor 20 minutes, NE processing 30 minutes；

2) PBS for adding 1mL/3.5cm dish sterilizings washs cell 2 times；

3) 150 μ l cell pyrolysis liquids are added in the washed Tissue Culture Dish of PBS and (protease are added before experiment Inhibitor and inhibitors of phosphatases), put and crack 30 minutes on ice, during which repeatedly shake Tissue Culture Dish, ensure to split Solve liquid uniform fold cell and crack abundant；

4) sufficient lysate check mark will be cracked in Tissue Culture Dish and is transferred to 1.5mL Eppendorf pipes；

5) 4 DEG C, 13000rpm high speed centrifugations 30 minutes；

6) collect supernatant and be transferred to new Eppendorf pipes, detect protein concentration with BCA methods, and adjust each sample The concentration of product is consistent；

7) albumen and loading buffer are mixed, 100 DEG C of metal baths boil sample 10 minutes, treat that albumen cools down After 4 DEG C, 13000rpm high speed centrifugations 15 minutes, albuminate loading, 120V, electrophoresis time 1 are taken Hour；

8) the prior NC films for preparing size suitable (energy coverage goal protein domain, film are more slightly larger than filter paper), filter paper, It is standby to be soaked in transferring film liquid；

9) the complete SDS PAGE glues of electrophoresis are taken out from glass plate, the unnecessary glue of upper and lower side is cut off, in Thermo In Fisher transferring films box transferring film " three is installed by negative pole to the order of positive pole sponge-filter paper-glue-film-filter paper-sponge Mingzhi "；

10) membrane-transferring device is correctly installed in electrophoresis/transferring film box, pours into transferring film liquid, 300mA constant currents transferring film 90 Minute；

11) take membrane-transferring device apart, take out transfer membrane, sandwich (prevents film surface temperature height after transferring film, rapidly immediately Kill) in preprepared TBST, 5 minute/time in TBST, washing is three times；

12) 5% skimmed milk power room temperature is closed 1 hour, and then 5 minute/time in TBST, is washed three times；

13) 1 is pressed using TBST:1000 dilution proportion primary antibodies, it is incubated at room temperature 1 hour, then in TBST 5 minutes/time, washing is three times；

14) 1 is pressed according to primary antibody source selection secondary antibody, TBST:2000 dilutions, incubation at room temperature 1 hour, then 5 minute/time in TBST, washing is three times；

15) ECL luminescent solutions are prepared, the NC films after washing are pressed from both sides out with clean tweezers and put preservative film, are added dropwise ECL luminescent solutions, luminescent solution is drained after 1 minute, covers preservative film；

16) according to 2 seconds, 10 seconds, 2 minutes, 10 minutes in darkroom, the time for exposure order of 30 minutes exposes Light, multiple film tablettings, and proper extension time for exposure also may be selected；

17) film after exposure is placed in automatic developing fixing in Kodak machines, until drying slice.

9.RT-PCR

Fluorescence real-time quantitative PCR experiment material and instrument

RNA is extracted from cell with Trizol methods, Trizol is purchased from Thermo Fisher companies (Cat No： 15596026), cDNA synthetic agent box comes from Takara companies primerscript 1st strand cDNA synthesis kit(Cat No：D6110), fluorescence real-time quantitative PCR kit is the SYBR of Takara companies premix ex taq(Cat No：DRR041s).Fluorescence real-time quantitative PCR instrument is ABI companies 7500Fast Real-time fluorescence quantitative PCR instrument.Primer is conventional design and closed by Suzhou Jin Weizhi bio tech ltd Into.

10.Trizol methods RNA is extracted

1) cell of 3.5cm culture dishes is cracked with 1mL Trizol reagents；

2) above-mentioned lysate is transferred in EP pipes, placed 5 minutes at 15-30 DEG C of room temperature；

3) in above-mentioned EP pipes, the amount for adding 0.2mL chloroforms by every 1mL Trizol adds chloroform, covers EP Lid, firmly shaken in hand 15 seconds, place 2-3 minutes at room temperature；

4) (2-8 DEG C) of 12000g is centrifuged 15 minutes；

5) take upper strata aqueous phase to be placed in new EP pipes, add the amount of 0.5mL isopropanols to add according to every 1mL Trizol Enter isopropanol, place 10 minutes at room temperature；

6) (2-8 DEG C) of 12000g is centrifuged 10 minutes；

7) supernatant is abandoned, waste liquid is blotted with blotting paper, adds the ethanol of 1mL 75% to be washed according to every 1mL Trizol Wash, whirlpool mixing, (2-8 DEG C) of 7500g is centrifuged 5 minutes, abandons supernatant；

8) blotting paper blots waste liquid, allows the RNA of precipitation to spontaneously dry at room temperature；

9) RNA precipitate is dissolved with 50 μ l RNase-free water；

10) RNA obtained with DNase I processing, 37 DEG C of water-baths 30 minutes.

11) 1 μ l stop solution (50mM EDTA), 65 DEG C of water-baths 10 minutes, after taking out are added Insert at once on ice, that is, the total serum IgE purified；

12) by institute in the primerscript 1st strand cDNA synthesis kit of Takara companies The specification of offer, select Oligo primers to carry out reverse transcription, obtain cDNA.

The PCR programs of NE acceptors are：94 DEG C of 2min (94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C 20 S, 30cycle), 72 DEG C of 10min.

The RT-PCR programs of P2 acceptors are：94 DEG C of 10min (95 DEG C of 25s, 60 DEG C of 30s, 72 DEG C 30s, 40cycle).

The humidification for the brown fat cell heat production that the ATP of embodiment 1 is induced NE

ATP wants to play a role, and necessarily have detected by the acceptor on brown fat cell film, inventor Gene expression abundance of the ATP acceptor (P2receptor) in brown fat cell, find ATP acceptors in brown Fat is there is the expression of more hypotypes, in P2X receptor subtypes, P2X1 and P2X4 express account for it is leading, In P2Y receptor subtypes, P2Y13 and P2Y14 expression accounts for leading, and other hypotype abundance are relatively low, but still may be used It can play an important role.

Inventor selects 10 μM of ATP and 0.1 μM of NE collective effect cell before.

By ATP and NE collective effects in brown fat cell, as a result find, ATP and ATP- γ S can show Write the brown fat cell heat production (Figure 10) of enhancing NE inductions.ATP is embodied in two to NE humidification Aspect：One, the cell number of reaction, in 158 cells of 5 experiments, the cell for having 95% is heat production (figure 10A), it is obviously improved compared with NE (norepinephrine) independent role, and ATP independent roles will not then draw The exothermic reaction (Figure 10 B) for sending out brown fat cell obvious；Two, the quantity of heat production of individual cells has obvious increasing Add, add 90%.The change (Figure 10 C) of quantity of heat production in the cell of heat production is compared, NE+ATP is significantly carried High the heat production amplitude of individual cells (P ＜ 0.01).

As a result show, heat production of the ATP to the NE brown fat cells induced significantly increases effect, it is seen that In vivo, when NE and ATP discharge jointly, the heat production efficiency of brown fat cell is very high.

The humidification for the brown fat cell heat production that the ATP synthetase inhibitors of embodiment 2 are induced NE with And the influence of membrane voltage

Rotenone (rotenone) is a kind of inhibitor of electron transport chain complexⅠ, it block electronics by Transmission of the NADH to CoQ.During experiment, 5 μM of addition rotenone 30 minutes, then adds 0.1 μ in advance M NE, as a result show, all cell heat production are all activated, but can not continue (Figure 18).Show NADH Be not activation heat production necessary to, but cell continue heat production needed for, it provide proton gradient be only Brown fat cell can endlessly carry out the energy source of heat production.

Oligomycin (oligomycin) is one of inhibitor of oxidative phosphorylation, the Fo portions with ATP synzyme Divide (part that film inner proton passes through) to combine, can specifically suppress the transport of proton, so also suppressing ATP's Synthesis and decomposition.

During experiment, while 10 μ g/mL oligomycin and 0.1 μM of NE are added, as a result as shown in figure 19. As a result show, each cell has a stronger heat production, and the lasting decline of membrane voltage of cell.To collect More cellular informatics as far as possible, centered on the visual field of time image, before NE with plus NE after, clap 3x3 respectively 9 visuals field, by the contrast of front and rear imaging, obtain the result of the membrane voltage change and heat production of cell. This time 9 visuals field of experiment share 172 cells, and each cell has exothermic reaction, and under membrane voltage Drop, it is seen that ATP synzyme plays a significant role really in the maintenance of mitochondrial membrane voltage.

The membrane voltage change of cell when the NE of embodiment 3 is induced

In the temp measuring method (method is referring to Chinese Patent Application No. 201410850735.3) of temperature sensitive dyes Mention, rhodamine 800 can reflect the change of membrane voltage, that is, temperature sensitivity as a reference dye The temp measuring method of dyestuff can also monitor the change of mitochondrial membrane voltage simultaneously except the change of detection temperature.

As a result such as Figure 17 A, shown in 17B.As a result show, mitochondrial membrane voltage has different when NE is handled Change, in 150 cells of five experiments, 55.6% cell is that membrane voltage declines, and membrane voltage Decline along with cell temperature increase.Among these cells, the cell for having 76% is heat production, Namely there are nearly 20% cell membrane potential increase and heat production.

As a result show, mitochondrial membrane voltage has slight rising, then declines or continue again to rise.This The slight rising of section and the increase of cell reduced coenzyme are consistent (Figure 14 F), are that cell metabolism strengthens As a result.

Under normal circumstances, the source of proton gradient is I, III, IV in the compound in electron transport chain, And when cell is in anoxic or membrane voltage declines, the function of synthesis ATP ATP synzyme can be used in Reversion, hydrolysising ATP pump proton, to maintain the normal membrane voltage of cell.

In addition, when with the addition of ATP synthetase inhibitors (such as oligomycin), it was observed that nearly all cell Mitochondrial membrane voltage depolarize.

The pH value change of cell when the NE of embodiment 4 is induced

The heat production of NE induction brown fat cells simultaneously, has it was observed that intracellular pH value changes with membrane voltage degree of depolarization There is positive correlation (Figure 17 C hollow dots), that is, the Cellular pH value depolarized can increase, the cell of hyperpolarization, PH can be reduced.

The cell that membrane voltage depolarising is mentioned in embodiment 3 is mostly heat production cell, therefore intracellular pH value increases Cell be mostly heat production cell.By extramitochondrial proton leak to Intramitochondrial when this is activated with UCP1 Conclusion is (proton concentration of endochylema, which reduces, causes pH value to raise) to match.Simultaneously, it was further observed that with 10 μ G/mL oligomycin all depolarizes (Figure 17 C solid dots) in the case of ATP synthesis enzyme levels, cell, and Intracellular pH value is mostly to increase.The result shows that the change of intracellular pH value can be used as brown fat cell heat production degree A Measure Indexes.

Intracellular ATP change when the NE of embodiment 5 is induced

AT1.03 is by the way that the epsilon subunit (about 14kDa) of bacterium FoF1-ATP synzyme is specific and ATP With reference to but not hydrolysising ATP, FoF1-ATP synthesis enzyme epsilon subunits and ATP cause huge conformation to become when combining Change, so that FRET (Sze et occur for the CFP and mVenus that are incorporated in epsilon subunit both ends Al.), when laser confocal microscope is taken pictures, with 458nm laser excitation CFP, eCFP is collected respectively With mVenus transmitting light, launch light (535nm-565nm) intensity and eCFP by calculating mVenus Launch the ratio of light (480nm-495nm) intensity, so as to the level of ATP in indicator cells.Plus line grain The ATP that the positioning signal clone of body can be measured in mitochondrial matrix into new plasmid mito-AT1.03 is dense Degree.

As a result as shown in figs.As a result showing, intracytoplasmic ATP declines rapidly when NE is handled, Intramitochondrial ATP contents also have different degrees of decline.On the brown fat mitochondria separated Experiment shows, when mitochondria is in uncoupling state, only a small amount of ATP generation (deficiency original 20%). Because every vital movement of cell is still continuing, ATP consumption is still continuing, so intracellular ATP Reduction is a complicated process.But intracellular ATP decline can activate the restriction enzyme phosphoric acid fruit of glycolytic cycle Sugared kinases, strengthens glycolysis, increases ATP supply.But the ATP levels of cell are constantly in afterwards Relatively low level, the sign not recovered, show there is the mechanism by NE activation persistently to disappear into the cell Consume intracellular ATP.

Calcium reaction intracellular when inducing the NE of embodiment 6

Calcium ion plays an important role as intracellular second messenger to intracellular metabolism.ATP and NE The increase of Calcium ion can be triggered.

Inventor measures the change (Figure 11) of intracellular calcium concentration with dyestuff fura2-AM.As a result show, Calcium ion level heterogeneity (Figure 11 A, 11F) in brown fat cell, when 0.1 μM of NE is handled, Calcium in all cell cytosols is all significantly increased and (n=54, tested three times) (Figure 11 A, 11B).This persistently increases The calcium added, intracellular a variety of metabolic activity can be adjusted.

The increase of calcium ion can strengthen respiration in mitochondria, so as to produce more ATP or Heat.And CoQ reducing condition can have influence on activation of the long chain fatty acids to UCP1 in mitochondria.It is former It is not easy to transfect for brown fat cell, therefore the cell indifference tested every time is imaged, can provides strong Evidence.As a result show, Intramitochondrial calcium has obvious increase (Figure 12), it is seen that the metabolism of cell increases By force.The activation of the acceptors of α 1 can cause the release of endocytoplasmic reticulum calcium, and experimental result is shown, cell endoplasm Net calcium release there is very big fluctuation (Figure 13), this be probably cause Calcium ion increase fluctuate the reason for it One.

Although the experiment of transfection is because the limitation of cell quantity and the uncertainty of cell can not judge whether Contain two kinds of cell of heat production and not heat production, but from the reaction of entirety still can be seen that NE processing when, The calcium of mitochondria is increased, and the calcium of endoplasmic reticulum is release.And notice a bit, whether line grain Calcium in body or endoplasmic reticulum is all not returned to initial state after change.

The change of cellular redox state when the NE of embodiment 7 is induced

When NE is handled, the either calcium ion in endochylema or Intramitochondrial calcium ion concentration has very Big increase, calcium ion have vital influence to intracellular metabolism.

NADH (NADH) and flavin adenine dinucleotide (FAD) (Waller et al.) two Individual coenzyme participates in intracellular important metabolic process：Glycolysis, tricarboxylic acid cycle and oxidative phosphorylation, with ATP Have direct relation.Their state of oxidation (NAD+/FAD) and reducing condition into the cell be present (NADH/FADH2), but only FAD and NADH have obvious fluorescence.Experiment using FAD with FAD+NADH fluorescence ratio represents the metabolism state of cell.

As a result showing, FAD and NADH has stronger fluorescence (Figure 14 A, 14B) in brown fat cell, and And when NE is stimulated, intracellular redox state has a greater change (Figure 14 D)：FAD fluorescent value Only slight rising, and NADH fluorescent value suppression ratio is more violent, represent intracellular when NE is stimulated Reduced coenzyme (NADH) has stronger consumption.In addition, in Figure 14 E, all cells add NE it There is being slightly increased for a section of reduction type coenzyme before this afterwards, followed by a large amount of consumption of reduction potential energy.

As a result show, after NE is added, intracellular metabolism is enhancing, generates more reduced coenzymes. And the redox state that can be seen that each cell starting from Figure 14 C and 14E is not quite similar, to NE The reaction of stimulation has by force and had weak (Figure 14 E, 14F).

The heat production of the brown fat cell of the NE of embodiment 8 inductions

Application of the 8.1 mitochondria thermometries in primary brown fat cell

The research of brown fat cell heat production is carried out using mitochondria thermometry, can be in unicellular water The data of substantial amounts of mitochondria heat production are obtained on flat, are provided so as to study the heat production for studying brown fat cell Good instrument.

Electron microscopic data shows there is substantial amounts of mitochondria in brown fat cell, therefore can be when body needs Carry out efficient heat production.Light-sensitive coloring agent rhodamine B methyl esters can be positioned at the mitochondria (figure of brown fat cell 2), and mainly by mitochondria in heat production in brown fat, can use herein photosensitive dye, rhodamine 800 and The ratio of the fluorescence intensity of rhodamine B methyl esters represents the change of cell temperature.And due to caused by mitochondria The transmission that heat will pass through heat gets to whole cell, therefore in fast-changing temperature survey, line The temperature change of plastochondria can only reacting cells temperature change trend, if by the use of the temperature change of mitochondria as The temperature change of cell, it can undoubtedly over-evaluate the overall temperature change of cell.Therefore, in measure brown cell During heat production, the analysis of quantitative and semi-quantitative, that is, heat production and the relative quantity of heat production of brown cell are only done, Without temperature change caused by quantification heat production.

The brown fat exothermic reaction that 8.2 0.1 μM of NE trigger

Primary brown fat cell is handled with 0.1 μM of NE, medicine can stay in always after agent-feeding treatment Observe in liquid.The cellular informatics more than phototoxicity and acquisition as far as possible during to reduce laser confocal imaging, not On the premise of influenceing experimental result subsequent treatment, inventor reduces the resolution ratio of imaging, therefore, it is impossible to obtain Clearly mitochondria is obtained to be imaged, but the change of fluorescence intensity can still detect, can also be anti-on the whole Answer the change (Fig. 3 F) of cell mitochondrial temperature.NE, and beta 3 receptor specific agonist CL316243 or Beta receptor wide spectrum agonist isoproterenol (Isoprenaline) is all only capable of causing the primary brown fat in part The exothermic reaction (Fig. 3) of fat cell.The heat production response of primary brown fat cell is not consistent, in 5 NE It is heat production (Fig. 3 A) that 150 cells, which only have 76% cell, in the experiment of stimulation, and test every time due to The problem of visual field is chosen, this numerical value can slightly have difference.Therefore it is being related to subsequent statistical heat production cell quantity Experiment when, all carry out multiple repetition parallel laboratory test.

For the ease of the unification of data, the imaging temperature of cell is 33 DEG C, it is worth mentioning at this point that, it is initially real Test when imaging temperature is set into 37 DEG C, it is dead after many cell heat production, prompt that cell is resistance to temperature to receive one Determine scope.Further, since the dynamic change speed of two dyestuffs can not possibly be completely the same, therefore, in NE That small cooling after addition is probably an illusion, it is also possible to it is anti-to reflect a real stimulation Should, because same phenomenon is also clearly visible with fluorescin temp measuring method.However, these do not influence finally The final temperature change of cell when stable.

The effect of 8.3 NE acceptors and inhibitor

The mRNA of primary brown fat cell is collected, enters performing PCR amplification, 9 kinds of different NE acceptors of discovery Subtype expression differs, and wherein α 1B only have the expression of trace, other several expression of receptor amount phases with alpha-2 receptor As high (Fig. 4).

SR 59230A in high concentration for wide spectrum NE acceptors inhibitor (Leblais et al., 2004), In the presence of 1 μM of SR 59230A, the brown fat cell exothermic reaction of 0.1 μM of NE initiation It can be totally constrained (Fig. 5), it is seen that the cell of heat production is strictly receptor-mediated by NE, is not due to The presence of NE medicines makes illusion caused by two kinds of dyestuff redistributions.Although SR 59230A inhibit cell Exothermic reaction, but P-HSL (Ser563) phosphorylation is not complete to suppress (Fig. 7), it is seen that only A small amount of P-HSL (Ser563) phosphorylation can not excite the exothermic reaction of brown fat cell.

In order to further verify the effect of acceptor, the inhibitor of the higher acceptor of known specificity have chosen： CGP-20712 (β1receptor inhibitor).By inhibitor preact in cell, then add 0.1 μM of NE thorn Swash, observe the exothermic reaction of cell.It was found that the cell of heat production significantly reduce (42%, n=180 cells, 5 experiments), and some cell temperatures start to reduce after 30 minutes, make the exothermic reaction of whole cell " bell " (Fig. 6 B, 6E) is presented in curve, shows in the brown fat cell of maturation, and β1receptor participates in Lasting cell exothermic reaction.Further immunoblot experiment is it is also seen that suppressing β1receptor can subtract Few P-HSL (Ser563) phosphorylation (Fig. 7), so as to prove that β1receptor take part in the thin of NE inductions really Born of the same parents' heat production.

The brown fat cell exothermic reaction that 8.4 10 μM of NE trigger

Since 0.1 μM of NE can not cause the exothermic reaction of whole cells, if be due to cell NE not Otherness with affinity expression of receptor causes the activation concentration for being not reaching to maximumNE concentration is increased, Really intracellular P-HSL (Ser563) phosphorylation degree (Fig. 8 A, 8B) can be increased.10 μM of NE can To be slightly increased the heat production amplitude of cell (Fig. 8 D).But in 110 cells of 3 experiments, have 81% cell is heat production, 76% no obvious difference (P=0.26) with 0.1 μM of NE.NE concentration increases Add 100 times, still all cells can not be made to have exothermic reaction.

UCP1 is expressed in the adipocyte of embodiment 9

Increase NE concentration, whole cells are not still entered into line activating heat production.Previously with respect to brown fat Research surveys oxygen demand all of fat cell heat production replaces difference with cAMP growing amount, this observation To the heat production heterogeneity of individual cells, if be due to that the primary brown fat cell of separation is mixed with white adipose Reason.The important molecule that brown fat cell is different from white adipose is exactly the uncoupling egg of mitochondria In vain：UCP1.The dyeing of cell is carried out with UCP1 antibody, finds have in the adipocyte of all separation UCP1 expression, cromoci are mitochondrial molecule mark albumen, show that the mitochondria of UCP1 antibody is special Property positioning (Fig. 9).

Mechanism of action of the ATP of embodiment 10 to NE humidifications

When inventor have detected NE with ATP collective effects, P-HSL (Ser563) Phosphorylation status, find NE and ATP collective effects increase P-HSL (Ser563) phosphorylation to reduce, this with brown fat cell The P2Y13 of height expression matches with P2Y14 acceptors, can because the two acceptors are mutually coupled with Gi albumen To suppress PKA function.Illustrate ATP to cracking that NE humidification is not by increasing ester acyl glycerine Come what is realized.

The change of redox state shows the change of endocellular metabolism state, and intracellular oxidation state represents FAD and NAD+ content is high, when meeting inducing cell produces excessive ROS, NE and ATP collective effect, carefully It is small (Figure 20 B) when the change of intracellular oxidation state is than NE independent roles, show that intracellular ROS yield is reduced, Because the heat production cell of ROS inductions will not also increase.

As shown in Figure 20 C, although ATP has obvious increase to the cell number of the NE heat production induced, It is still to have nearly 20% cell membrane potential to increase and cell heat production, if ATP synzyme serves weight wherein The effect wanted, then the effect of Extracellular ATP is exactly that the function reversal effect for making ATP synzyme is inhibited, Or total long-chain fat acid concentration increase, make UCP1 suppression again become difficult, now long-chain fat The increase of acid can only be phosphatidase PLA2 effect, between being gone by the change of pH in endochylema and calcium ion Connect the activated state for understanding PLA2.

Bibliography

1.Imamura,H.,Nhat,K.P.H.,Togawa,H.,Saito,K.,Iino,R., Kato-Yamada,Y.,Nagai,T.,and Noji,H.(2009).Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proceedings of the National Academy of Sciences 106,15651-15656.

2.Imamura,H.,Nhat,K.P.H.,Togawa,H.,Saito,K.,Iino,R., Kato-Yamada,Y.,Nagai,T.,and Noji,H.(2009).Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proceedings of the National Academy of Sciences 106,15651-15656.

3.Palmer,A.E.,Giacomello,M.,Kortemme,T.,Hires,S.A.,Lev-Ram, V.,Baker,D.,and Tsien,R.Y.(2006).Ca 2+indicators based on computationally redesigned calmodulin-peptide pairs.Chemistry& biology 13,521-530.

4.Palmer,A.E.,Jin,C.,Reed,J.C.,and Tsien,R.Y.(2004). Bcl-2-mediated alterations in endoplasmic reticulum Ca2+analyzed with an improved genetically encoded fluorescent sensor.P Natl Acad Sci USA 101,17404-17409.

All it is incorporated as referring in this application in all documents that the present invention refers to, just as each document It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen Please appended claims limited range.

Claims

1. a kind of purposes of heat production enhancing compound, it is characterised in that for preparing a preparation or composition, institute State preparation or composition is used to strengthen the heat production efficiency that NE classes compound induces brown fat cell, wherein, it is described Heat production enhancing compound is selected from the group：ATP classes compound, mitochondrial complex V/ATP synthetase inhibitors or It is combined.

2. purposes as claimed in claim 1, it is characterised in that the ATP classes compound is selected from the group：ATP、 ATP- γ S, BzATP, α, β-methylene adenosine triphosphate, 2- methylthioadenosines triphosphoric acid, ADP, UTP, UDP, MRS2690, UDPG, UDP- galactolipins or its combination.

3. purposes as claimed in claim 1, it is characterised in that the mitochondrial complex V/ATP synthesis Enzyme inhibitor is selected from the group：Oligomycin, polygodial, orthovanadate or its combination.

4. purposes as claimed in claim 1, it is characterised in that the preparation or composition also include NE Class compound.

A kind of 5. method of the enhancing Into The Thermogenesis In Mammals of external non-therapeutic, it is characterised in that including step Suddenly：

6. method as claimed in claim 5, it is characterised in that the NE classes compound increases with the heat production The mol ratio for strengthening compound is 1-100：10-1000, it is preferred that 1-5：50-500, more preferably, 1-2： 100-200。

7. method as claimed in claim 5, it is characterised in that the effect of the heat production enhancing compound is dense Spend for 0.1-100 μ g/mL, it is preferred that 1-50 μ g/mL, more preferably, 5-20 μ g/mL.

8. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition includes：

(i) NE classes compound；

(iii) pharmaceutically acceptable carrier.

9. a kind of medicine box, it is characterised in that the medicine box includes：

(a) the first preparation containing NE class compounds；

(c) specification.

A kind of 10. method for the compound for screening enhancing brown fat cell heat production, it is characterised in that including step Suddenly：