WO2017039096A1 - Composition pharmaceutique pour la prévention ou le traitement de troubles métaboliques, contenant du sphingosine-1-phosphate ou un matériau augmentant l'expression de sphk2 - Google Patents

Composition pharmaceutique pour la prévention ou le traitement de troubles métaboliques, contenant du sphingosine-1-phosphate ou un matériau augmentant l'expression de sphk2 Download PDF

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WO2017039096A1
WO2017039096A1 PCT/KR2016/002410 KR2016002410W WO2017039096A1 WO 2017039096 A1 WO2017039096 A1 WO 2017039096A1 KR 2016002410 W KR2016002410 W KR 2016002410W WO 2017039096 A1 WO2017039096 A1 WO 2017039096A1
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sphk2
sphingosine
phosphate
expression
liver
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PCT/KR2016/002410
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Korean (ko)
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박태식
심순미
이수연
맹효진
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가천대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/68—Sphingolipids, e.g. ceramides, cerebrosides, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/133—Amines having hydroxy groups, e.g. sphingosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating metabolic dysfunction, and more particularly, any one selected from the group consisting of sphingosine 1-phosphate, a synergist of sphingosine 1-phosphate, and a substance that increases the expression of Sphk2. It relates to a pharmaceutical composition for preventing or treating metabolic dysfunction comprising a.
  • Non-Patent Documents 1-3 Elevated plasma free fatty acids (FFAs) lead to abnormal fatty acid (FA) absorption in the liver, adipose tissue, and heart, which are responsible for fatty liver, inflammation, and cardiac dysfunction, respectively (Non-Patent Document 4-6). ). Increased tissue uptake of FFA under obese conditions leads to the synthesis of triglyceride (TG), phospholipids and sphingolipids. In this process, synthesis of specific lipid metabolites is known to provide signaling molecules that regulate metabolic function systems (Non Patent Literature 7). Lipid-mediated regulation of the cell's insulin signaling system is associated with the activation of serine / threonine kinase and leads to the progression of endogenous insulin activity in insulin-sensitive tissues (Non Patent Literature 8-10).
  • Non-Patent Document 11 Endoplasmic reticulum (ER) stress is induced by various cellular damages including glucose depletion, inflammatory conditions, and disruption of calcium homeostasis.
  • ER Endoplasmic reticulum
  • URR unfolded protein response
  • UPR releases GRP78 from ER, and protein kinase RNA-like ER associated kinase (PERK) pathway, inositol-requiring protein 1 ⁇ (IRE1 ⁇ ) pathway, and transcription It begins with the activation of three signaling systems of the activating transcription factor 6 (ATF6) pathway (Non-Patent Document 14).
  • the PERK pathway is activated in diet-induced obese (DIO) mice, while the chemical inducers that induce acute ER stress, such as tunicamycin and thapsigargin, are described above. By inducing all of the canine UPR signaling pathways, it suggests that these pathways play distinct roles under different conditions of physiological stress (Non-Patent Documents 15-17).
  • Such transcription factors are involved in the expression of UPR genes, including chaperone and lipid producing genes.
  • SphK Sphingosine kinase catalyzes the phosphorylation of sphingosine to synthesize sphingosine 1-phosphate (S1P), a lysophospholipid (non-patent document 18).
  • SphK is a 49 kDa protein that has been purified from rat kidney and identified. As a result of cloning, the SphK contains Sphk1a and Sphk1b (Non-Patent Documents 19 and 20).
  • Sphk2 the second isoform, has high homology with Sphk1, and both isoforms have five conserved domains found in lipid kinases (Non-Patent Document 19).
  • Sphk1 is highly expressed in lung, spleen, kidney, and blood, while Sphk2 is mainly expressed in liver, kidney, and heart (Non-Patent Documents 20 and 21).
  • S1P levels are precisely regulated by synthesis of SphK catalyst, irreversible degradation of S1P lyase and generation of sphingosine by dephosphorylation by S1P phosphatase (Non-Patent Document 22).
  • S1P is a ligand for the specific G protein receptor (S1P 1 -S1P 5 ) family. S1P functions through secretion and binding to the S1P G-protein receptor, a process referred to as "inside-out" signaling (Non-Patent Document 22).
  • Sphk1 is pro-apoptotic, whereas Sphk2 has an anti-apoptotic role on the contrary (Non-Patent Document 23).
  • the present inventors studied to determine whether ER stress induced Sphk2 expression and raised liver S1P.
  • Sphk2 was upregulated by acute ER stress, and Sphk2 with increased expression in liver was not associated with proximal insulin signaling.
  • AKT also referred to as serine / threonine-specific protein kinase (PKB, AKT)
  • PKT serine / threonine-specific protein kinase
  • the present invention provides a pharmaceutical composition and food composition for the prevention or treatment of metabolic dysfunction, including any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate synergist, and a substance that increases the expression of Sphk2. And a cosmetic composition and a method for screening a substance for preventing or treating metabolic dysfunction using the above mechanism.
  • a pharmaceutical composition for preventing or treating metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate synergist, and a substance that increases the expression of Sphk2. This is provided.
  • the Sphk2 can be liver specific Sphk2.
  • the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia and obesity, the fatty liver may be non-alcoholic fatty liver, and the dyslipidemia is hyperlipidemia or hypertriglyceridemia Can be.
  • the substance that raises the expression of Sphk2 may be DNA or RNA.
  • the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or additives.
  • a food composition for improving metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, a synergist of sphingosine 1-phosphate, and a substance that increases the expression of Sphk2.
  • the food composition may be any one selected from the group consisting of dietary supplements, nutraceuticals and food additives.
  • a cosmetic composition for improving metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, a synergist of sphingosine 1-phosphate, and a substance that increases the expression of Sphk2 Is provided.
  • step (a) preparing a cell or tissue of a high fat dietary animal; (b) contacting the test substance with the cell or tissue of step (a); (c) measuring the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in the cells or tissues of step (b); And (d) selecting a test substance which exhibits an increase in the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein compared to the control group which has not been treated with the test substance.
  • a screening method of is provided.
  • Sphk2 overexpression in the liver and subsequent increase of S1P activate oxidative gene upregulation and fatty acid oxidation. It has been shown to reduce hepatic fat accumulation and to alleviate ER stress-mediated abnormalities by indicating increased insulin sensitivity and amelioration of insulin resistance by activation of the insulin signaling system through an increase in pAKT.
  • the composition comprising any one selected from the group consisting of sphingosine 1-phosphate of the present invention, a synergist of sphingosine 1-phosphate and a substance that raises the expression of Sphk2 may be used for metabolic dysfunction, in particular, non-alcoholic fatty liver and the like. It can be usefully used as a pharmaceutical composition, food composition and cosmetic composition for the prevention or treatment of dyslipidemia and obesity, such as fatty liver, hyperlipidemia or hypertriglyceridemia, the screening method using the mechanism for the prevention or treatment of metabolic dysfunction It can be usefully used as a screening method for selecting a substance.
  • CerS2-5 (Ceramide synthase2-5, ceramide synthase2-5); AcCer (Acid ceramidase, acid ceramidase); Acer 1-3 (Alkaline ceramidase 1-3, alkaline ceramidase 1-3); Activating transcription factor 4 (ATF4); Activating transcription factor 6 (ATF6); CHOP (C / EBP homologous protein, C / EBP homologous protein); NCer (neutral ceramidase, neutral ceramidase); Smpd1 (acid sphingomyelinase); Smpd2 (neutral sphingomyelinase 1, neutral sphingomyelinase 1); Smpd3 (neutral sphingomyelinase 2, neutral sphingomyelinase 2); Sphk1-2 (sphingosine kinase 1-2, sphingosine kinase 1-2); sXBP1 (spliced X
  • FIG. 5 shows Sphk2 expression after co-transfection of a reporter construct comprising a Sphk2 promoter with ATF4, sXBP1 or CHOP, respectively.
  • FIG. Specifically, the Sphk2 reporter construct and pcDNA 3.0 (0.2 ⁇ g or 0.4 ⁇ g) comprising ATF4, sXBP1 or CHOP were co-transfected into HepG2 cells. Promoter activity was measured by luciferase assay. Three independent experiments were performed to show the results (A) (n 5, mean ⁇ SEM, * p ⁇ 0.05 vs. control blank pcDNA3.0 control, #p ⁇ 0.05 vs. 0.2 ⁇ g transfected group).
  • Figure 6 shows infection of primary mouse hepatocytes with adenovirus expressing human Sphk2 (AdSphk2), followed by expression levels of each protein (A), sphingoid base level (B), ceramide (Cer) level (C) and This shows the sphingomyelin (SM) level (D), and the expression level (E) of each protein after HNMPA administration to Sphk2 overexpressing cells.
  • AdSphk2 adenovirus expressing human Sphk2
  • FIG. 8 shows that Sphk2 overexpression in the liver ceramide (A), dihydroceramide (B), sphinginine (C), sphingosine (C), sphingosine 1-phosphate (C), sphingomyelin (D), glucose
  • silceramide E
  • cholesterol / triglyceride F
  • WT C57Bl / 6 mice fed high fat diet (60 kcal% fat) for 4 weeks were infected with AdGFP or AdSphk2 (1 ⁇ 10 9 PFU / ml) by tail vein injection.
  • Mouse livers were isolated 14 days after injection.
  • FIG. 10 shows plasma glucose (A), plasma insulin levels (B), plasma glucose (C) following insulin infusion, the expression level of glucose neosynthesis gene (D), and AKT phosphorylation levels in skeletal muscle / fatty tissue in Sphk2 overexpressing mice (FIG. It is the result of measuring E).
  • WT C57Bl / 6 mice fed high fat diet (60 kcal% fat) for 4 weeks were injected with AdGFP or AdSphk2 (1 ⁇ 10 9 ) into the tail vein. Experiments were performed 7 days after adenovirus injection. Mice fasted for 16 hours were injected with 1 g / kg body weight of glucose and plasma glucose was measured at the indicated time (A). Plasma insulin levels were measured during the glucose tolerance test (B).
  • the present invention provides a pharmaceutical composition for preventing or treating metabolic dysfunction, including any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate synergist, and a substance that raises the expression of Sphk2.
  • Sphk2 may be liver specific Sphk2, but is not limited thereto.
  • the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia and obesity, the fatty liver may be non-alcoholic fatty liver, and the dyslipidemia is hyperlipidemia or hypertriglyceridemia Can be.
  • the substance that raises the expression of Sphk2 may be DNA or RNA.
  • the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or additives.
  • the pharmaceutical compositions of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. .
  • the pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. Also included are diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like.
  • Oral solid preparations include tablets, pills, powders, granules, capsules and the like, which solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, and the like, and may include lubricants such as magnesium stearate, talc, and the like.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include water, diluents such as liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like.
  • Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl. Injectable esters such as oleate and the like.
  • aqueous solvents such as olive oil, and ethyl.
  • Injectable esters such as oleate and the like.
  • As the base of the suppository witepsol, macrogol, tween, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Dosages of sphingosine 1-phosphate, sphingosine 1-phosphate synergists and substances that increase the expression of Sphk2 contained in the pharmaceutical composition of the present invention may be determined by the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and Depending on the time period, it may be appropriately selected by those skilled in the art.
  • the sphingosine 1-phosphate, the synergist of sphingosine 1-phosphate, and the substance which raises the expression of Sphk2 are 0.0001 to 1000 mg / kg per day, preferably at a dose of 0.01 to 1000 mg / kg. It may be administered, the administration may be administered once or several times a day.
  • the pharmaceutical composition of the present invention may include 0.001 to 50% by weight of the sphingosine 1-phosphate, the synergist of sphingosine 1-phosphate, and the substance that increases the expression of Sphk2 with respect to the total weight of the composition.
  • the pharmaceutical composition of the present invention may be used in various routes in mammals such as rats, mice, livestock, humans, for example, oral, abdominal, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. Can be administered by
  • a food composition for improving metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, sphingosine 1-phosphate synergist, and a substance that increases the expression of Sphk2;
  • a cosmetic composition is provided.
  • the food composition may be any one selected from the group consisting of dietary supplements, nutraceuticals and food additives.
  • Sphk2 may be liver specific Sphk2, but is not limited thereto.
  • the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia and obesity, the fatty liver may be non-alcoholic fatty liver, and the dyslipidemia is hyperlipidemia or hypertriglyceridemia Can be.
  • the substance that raises the expression of Sphk2 may be DNA or RNA.
  • a cosmetic composition for improving metabolic dysfunction comprising any one selected from the group consisting of sphingosine 1-phosphate, a synergist of sphingosine 1-phosphate, and a substance that increases the expression of Sphk2 Is provided.
  • step (a) preparing a cell or tissue of a high fat dietary animal; (b) contacting the test substance with the cell or tissue of step (a); (c) measuring the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein in the cells or tissues of step (b); And (d) selecting a test substance which exhibits an increase in the concentration of sphingosine 1-phosphate or the expression level of Sphk2 protein compared to the control group which has not been treated with the test substance.
  • a screening method of is provided.
  • Sphk2 may be liver specific Sphk2, but is not limited thereto.
  • the metabolic dysfunction may be any one selected from the group consisting of fatty liver, dyslipidemia and obesity, the fatty liver may be non-alcoholic fatty liver, and the dyslipidemia is hyperlipidemia or hypertriglyceridemia Can be.
  • the test substance may be DNA or RNA.
  • Ceramide (C14: 0, C16: 0, C18: 0, C18: 1, C20: 0, C24: 0, C24: 1), Sphingomyelin (SM 16: 0, C18: 0, C18: 1), Sping Gossine (SO), sphinginine (SA), sphingosine 1-phosphate (S1P), and glucosyl ceramide (C16: 0, C18: 0, C18: 1, C24: 1) are used by Avanti Polar Lipids. Lipids, Alabaster, AL). Acetonitrile, methanol and chloroform were purchased from Fisher Scientific, Pittsburgh, Pa. Insulin was purchased from Eli Lilly, Humulin, and tunicamycin was purchased from Sigma-Aldrich, St. Louis, Mo.
  • HNMPA- (AM) 3 was purchased from Abcam, Cambridge, Mass.
  • K145 was purchased from Sigma-Aldrich, St. Louis, Mo.
  • ATF4-specific siRNA and control siRNA were purchased from Qiagen (Qiagen, Boston, Mass.).
  • mice Eight-week-old male C57Bl / 6 mice were purchased from Charles River Laboratories and raised freely with water and diet at a pathogen-free animal breeding facility (usually diet: Lab Diet Inc. Picolab rodent diet 20, high fat diet: 60 kcal% fat diet for 4 weeks; D12492 by Research Diet Inc.).
  • Recombinant adenovirus (1 ⁇ 10 9 PFU) was injected into the tail vein.
  • mice were fasted for 16 hours, and then glucose (2 g / kg body weight) was injected intraperitoneally (Non-Patent Document 41).
  • insulin performance tests mice were fasted for 4 hours and then insulin (1 Unit / kg body weight) was injected intraperitoneally.
  • insulin 0.5 unit / kg body weight
  • Tunicamycin 2.5 ⁇ g / g body weight, 2 hours
  • lipopolysaccharide LPS, 3 ⁇ g / g body weight, 8 hours
  • mice were fasted for 6 hours and sacrificed to collect blood and each tissue separately.
  • HepG2 cells were purchased from the American Type Culture Collection, Manassas, VA. Cells were incubated at 37 ° C., 5% CO 2 conditions in DMEM medium supplemented with 10% fetal bovine serum, 10 units / ml penicillin and 10 ⁇ g / ml streptomycin. Five MOIs (Multiplicity of Infection) of GFP and Sphk2 adenovirus were infected with HepG2 cells for 24 hours. Cells were harvested after treatment with insulin (50 nM) for 10 minutes. Another cell was treated with DMSO or HNMPA- (AM) 3 (10 uM) for 6 hours followed by insulin for 10 minutes. After adenovirus infection or transfection of the overexpression vector, cells were cultured in serum-free medium without insulin for 16 hours to measure expression and insulin effect.
  • DMSO or HNMPA- (AM) 3 (10 uM
  • Non-patent Document 24 Eight weeks-old wild type (WT) C57BL6 male mice were anesthetized and livers were isolated.
  • Primary hepatocytes were prepared according to the method described in the prior document (Non-patent Document 24) as follows. Cells were cultured in M199 medium supplemented with 10% FBS, 10 units / ml penicillin, 10 ⁇ g / ml streptomycin, and 10 nM dexamethasone (Welgene, Korea). After cells were attached, cells were infected with each adenovirus for 16-24 hours. In addition, cells were maintained in serum-free medium for one day and then treated with tunicamycin (1.25 ⁇ g / ml) for a defined time.
  • control and ATF4 siRNA oligomers were transfected with lipofectamine (lipofectamine, Invitrogen, Carlsbad, Calif.). After 48 hours of incubation, tunicamycin (1.25 ⁇ g / ml) was incubated in serum free.
  • the promoter sequence of Sphk2 (-2050 / -1) was amplified by PCR from mouse genomic DNA and inserted into pGL3 base vector (Promega) to prepare pSphk2-pGL3 reporter construct.
  • pcDNA3-flag-ATF4 and pcDNA-flag-sXBP1 were provided by fellow researchers (Cho, Sung Hoi, Ph.D., Korea University, Seoul, Korea).
  • Recombinant adenovirus was produced using a vector system (AdEasy Adenoviral Vector System, Stratagene, La Jolla, CA, USA) and a vector (pAdTrack CMV vector) (Non-Patent Document 25).
  • Mouse Sphk2 cDNA was cloned into AdTrack CMV vector and recombined using cells (BJ5183-AD-1 Electroporation Competent Cells, Stratagene, Calif.). After confirming recombination, the total adenovirus genome was isolated and transfected into HEK293 cells. The adenovirus was purified after 1 day dialysis in CsCl gradient gradient centrifugation and 4 ° C., sucrose buffer (10 mM Tris-HCl pH8.0, 2 mM MgCl 2, 4% sucrose). Purified adenovirus was stored at -80 ° C until use (non-patent document 42). As a control, adenoviruses expressing green fluorescent protein were prepared and used by the method described above.
  • HepG2 cells (4 ⁇ 10 5 cells / ml) are 80-90% confluent in 10% FBS, 10 units / ml penicillin, and 10 ⁇ g / ml streptomycin supplemented culture medium (Ham's F12 medium) Incubated in 24-well culture plates until.
  • transfection reagent FuGENE® HD transfection reagent, Roche, Mannheim, Germany
  • cells were prepared using the pSphk2-pGL3 reporter construct; And ER stress marker cloning vectors comprising ATF4, ATF6 or sXBP1-pcDNA3.0, respectively.
  • Transfection efficiency was standardized using pTK-RL, the expression vector for Renilla luciferase.
  • Non-Patent Document 26 Total cell proteins were lysed with cell lysis buffer containing 10 mM Tris, pH 7.8, 1 mM EDTA, 150 mM NaCl, phosphatase inhibitors and protease inhibitors. Immunoblotting was performed using 30 ⁇ g of protein according to the method described in the prior document (Non-Patent Document 26).
  • ⁇ -actin AKT, phospho-AKT (Ser473), insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2), phosphor-tyrosine (Cell Signaling Technology, Danvers, MA ), Sphk2, glucose 6-phosphatase (G6Pase), PEPCK, ATF4, peroxisome proliferation activated receptor ⁇ (PPAR ⁇ ), carnithine palmitoyltransferase 1 (CPT1), acyl CoA oxidase 1, Abcam, Cambridge, MA) And primary antibodies against XBP-1 (Santa Cruz Biotechnology, Santa Cruz, Calif.) Were used. Blots were developed with chemiluminescent substrates (Millipore, Bilerica, CA) and detected with a fluorescence image analyzer (LAS4000 luminescent image analyzer, Fujifilm, Japan).
  • Ceramide (C14: 0, C16: 0, C18: 0, C18: 1, C20: 0, C24: 0, C24: 1), sphinginine, sphingosine, S1P, SM (C16: 0, C18: 0, C18: 1) and glucosylceramide (C16: 0, C18: 0, C18: 1, C24: 1) were separated by HPLC using a C18 column (XTerra C18, 3.5 um, 2.1 * 50 mm), prior art (non-patent) A modification was applied based on Document 43) to ionize in a positive electrospray ionization mode.
  • MRM Multiple reaction monitoring
  • Plasma and liver triglycerides, cholesterol, HDL, LDL, NEFA were measured with a colorimetric kit (Wako, Wako Pure Chemical Industries, Ltd, Japan). Insulin was measured using Mouse Insulin ELISA kits (ALPCO). ALT and AST activity was measured with an activity assay kit (Sigma Aldrich). Plasma ketone bodies ( ⁇ -hydroxybutyrate) were measured by colorimetric assay kit (Cayman chemical, Ann Arbor, MI).
  • Oxygen consumption rate was measured using a measuring instrument (extracellular Flux Analyzer, XF-24, Seahorse Biosciences, North Billerica, MA, USA). Primary mouse hepatocytes were seeded in 24-well plates (2 ⁇ 10 4 / plate) and incubated overnight in M199 medium with 10% FBS. The cells were washed with medium and then infected with adenoviruses containing GFP or Sphk2 and then incubated for 24 hours.
  • OCR Oxygen consumption rate
  • livers were separated and fixed for 24 hours in 10% buffered formalin or frozen in OCT insert medium. 5 ⁇ m sections of the liver inserted into paraffin were stained with hematoxylin and eosin (Sigma-Aldrich). Frozen sections of the liver were stained with oil red O. Images were obtained directly using the slide scanner.
  • ER stress activates hepatic lipid biosynthesis by upregulating major lipid producing genes such as diagylglycerol acyltransferase 2 (DGAT2), sterol responsive element binding protein 1c (SREBP1c), and LIPIN2 (Non-Patent Document 27).
  • DGAT2 diagylglycerol acyltransferase 2
  • SREBP1c sterol responsive element binding protein 1c
  • LIPIN2 Non-Patent Document 27.
  • Sphk2 expression is differentially adjustable
  • HFD feeding Long-term HFD feeding (weeks 8 and 12 of HFD feeding, see Figures 3 (A) and 3 (B)) did not change the UPR mediator, whereas liver sXBP-1 activation was found in 4 weeks HFD feeding mice. , ATF6 did not change and expression of ATF4 was inhibited (see FIGS. 2 (A) and 2 (B)). HFD raises ceramide and sphingomyelin (SM) levels in plasma and liver, but S1P has been shown to decrease (no statistical significance, see FIG. 4).
  • SM sphingomyelin
  • LPS lipopolysaccharide
  • adenovirus overexpression of ATF4 in primary mouse hepatocytes elevated Sphk2 protein levels but did not show any change in sXBP1 overexpression (see FIG. 5 (B)).
  • ATF4 siRNA was transfected. Downregulation of ATF4 resulted in inhibition of Sphk2 mRNA and protein levels (see FIGS. 5 (C) and 5 (D)). From these results, it was confirmed that Sphk2 is transcriptionally regulated by ATF4.
  • Non-Patent Documents 28 and 29 The effects of ceramide and S1P on the regulation of the insulin signaling pathway have been reported (Non-Patent Documents 28 and 29). S1P, a product of Sphk, is known to improve glucose homeostasis in diabetic animal models (Non-Patent Document 29). To determine whether elevated Sphk2 expression regulates the ER stress-dependent signaling pathway, an adenovirus expressing human Sphk2 (AdSphk2) was constructed and used for infection of primary mouse hepatocytes.
  • AdSphk2 adenovirus expressing human Sphk2
  • Non-Patent Document 29 phosphorylation of AKT was increased in a gene-concentration dependent manner by AdSphk2 infection (see FIG. 6 (A)) (Non-Patent Document 29).
  • pIRS1 or pIRS2 levels there was no change in pIRS1 or pIRS2 levels, so increased pAKT was not associated with IRS1 or IRS2, the proximal insulin signaling intermediate protein.
  • increased pAKT appeared to be independent of tyrosine phosphorylation of IRS.
  • Sphk2 expression did not alter sphingolipid metabolites such as sphinginine (SA), sphingosine (SO), ceramide (Cer) and sphingomyelin (SM), but only elevated cellular levels of S1P (FIG. 6 (B) to 6 (D)).
  • AdSphk2 was injected into the tail vein of HFD fed WT mice for 4 weeks. Delivery of a recombinant adenovirus is known to lead to preferential targeting of the transgene to the liver (Non-Patent Document 30).
  • Wild-type C57bl6 / J mice fed high fat diet (60 kcal% fat) for 4 weeks were injected with tail vein AdGFP or AdSphk2 (1 ⁇ 10 9 PFU). Plasma was isolated at 14 days post-injection to determine hematological parameters and the results are shown in Table 1 (n 8, mean ⁇ SEM. * P ⁇ 0.05 vs AdGFP-injected mice).
  • liver enzyme levels such as ALT indicates that liver function is improved.
  • Sphk2 overexpression reduced the levels of ceramide (A), dihydroceramide (B), sphingosine (C), sphingomyelin (D), and glucosyl ceramide (E) in plasma , Sphingosine 1-phosphate level (C) was increased.
  • Sphk2 overexpression has been shown to raise S1P in the liver but decrease ceramide, SM, glucosyl ceramide, cholesterol and triglycerides (triglycerides, TG). Unlike in the case of plasma, the result of a markedly reduced TG in the liver suggests that synthesis or oxidation is regulated by Sphk2. From the above lipid profile results, hepatic Sphk2 expression can be seen to affect the regulation of lipid metabolism in HFD fed mice.
  • AKT phosphorylation was measured in skeletal muscle and adipose tissue to determine whether non-liver tissue was associated with improved glucose or endothelial activity, but no changes in pAKT levels were identified (FIGS. 10 (E) and 10 (F)). Reference).
  • ER is the main cellular organ responsible for protein maturation and mercury into other cell compartments. Accumulation of unfolded protein acts as a stress on ER, which is associated with metabolic dysfunction and progression of inflammatory diseases (Non-Patent Document 32). Nutrient ingestion and infection-mediated inflammatory responses are initiated such that UPR maintains ER homeostasis as a physiological condition that stresses ER structure and function. If ER stress conditions persist, lipid homeostasis is disturbed and signal transduction of cells such as diacylglycerol (DAG) and FFA disrupts cellular signaling.
  • DAG diacylglycerol
  • ER stress-induced UPR is mediated by three ER-membrane associated proteins, PERK, IRE1 ⁇ , and ATF6.
  • ER stress-activated transcription factors such as ATF4, XBP1 and ATF6 induce various chaperones and correct the functional future resulting from the accumulation of unfolded proteins.
  • Excessive UPR signaling leads to metabolic disturbances including obesity and fatty liver.
  • Each UPR signaling pathway has a different function in metabolic regulation. For example, XBP1 deficiency results in the inhibition of new liver lipid biosynthesis, leading to reduced plasma TG, cholesterol and free fatty acids (Non-Patent Document 33).
  • ATF4 is activated in DIO mice to activate lipid production, and PERK-eIF2 ⁇ (-ATF4) is involved in the activation of inflammatory NF- ⁇ B (Non Patent Literatures 34 and 35). Although there is confusion in this UPR classification, metabolic dysregulation and inflammation are major categories of the UPR pathway involved in the stress-signaling pathway.
  • Non-oxidative FA pathways are involved in the biosynthesis of sphingolipids, and their metabolism leads to the biosynthesis of various bioactive lipid metabolites, including ceramide, SO and S1P.
  • Such sphingolipid metabolites are important signal transmitters in metabolic regulation (Non-Patent Documents 36 and 37).
  • Infusion of saturated FA results in an increase in ceramide levels in muscle and liver, and leads to terminal insulin ability (Non Patent Literature 9).
  • Pharmacological and genetic inhibition of new ceramide biosynthesis has been shown to improve glucose sensitivity and insulin response in myriosin-treated diet-induced obesity (DIO) mice or heterozygous Sptlc2-deficient mice (non-patent) Document 38, 39).
  • Non-Patent Document 29 shows the relevance of ceramide and S1P in liver glucose / lipid metabolism to nutritional status.
  • each UPR pathway selectively regulates the cause-centric target of stress signaling.
  • inflammatory ER stress such as that caused by LPS
  • hepatocytes may require an urgent response through the combined effects of the inflammatory response and ER stress mainly by Sphk2 induction via ATF4.
  • Sphk2 induction While both XBP1 and ATF4 account for liver lipid metabolism, S1P biosynthesis is regulated at least differently in the liver.
  • Sphk2 expression is believed to be regulated differently by UPR transcription factors under different pathophysiological conditions. In order to elucidate the role of Sphk2 and its product S1P in metabolic / inflammatory regulation, further studies on the stress signaling pathways associated with ATF4-mediated Sphk2 upregulation are needed.
  • adenoviral Sphk2 upregulation did not alter basal glucose levels in vivo, but only altered insulin response by activating signaling pathways via increased pAKT. Independently, plasma TG remained unchanged, while liver TG accumulation was reduced by Sphk2 expression. The decrease in hepatic TG was found to be due to upregulation of FA oxidative genes and increased FA oxidation. Sphk2 expression is an important regulator of FA oxidation, indicating that AdSphk2-infected hepatocytes increased oxygen consumption and elevated plasma ketone bodies. However, from the results that pharmacological Sphk2 inhibition did not alter glucose / FA metabolism, it can be seen that there may be a complementary mechanism by liver Sphk2. The Sphk2-mediated activation mechanism of liver FA oxidation and the role of S1P require further study.
  • ER stress-mediated Sphk2 upregulation occurs via ATF4 activation under inflammatory conditions and improves liver glucose and lipid abnormalities. Further elucidation is needed regarding the mechanism by which ER stress-mediated Sphk2 activation improves glucose sensitivity and FA oxidation.
  • the UPR signaling pathway may be an adaptive process for mitigating the stress state of the ER by FA oxidation activation.
  • Sphk2 upregulation leads to a decrease in fat by increased FA oxidation.
  • Sphk2-mediated activation of AKT improved insulin signaling under pathophysiological conditions.
  • Sphk2 is regulated by a UPR sensor that depends on the cause of ER stress induction and acts as a molecular messenger to relieve hepatic ER stress, maintaining glucose and lipid homeostasis.

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Abstract

La présente invention concerne : une composition pharmaceutique, une composition alimentaire et une composition cosmétique pour la prévention ou le traitement de troubles métaboliques, contenant un composé quelconque choisi dans le groupe constitué par le sphingosine-1-phosphate, un activateur du sphingosine-1-phosphate et un matériau augmentant l'expression de Sphk2 ; et un procédé de criblage d'un matériau pour la prévention ou le traitement de troubles métaboliques utilisant le mécanisme. La composition pharmaceutique, la composition alimentaire et la composition cosmétique de la présente invention peuvent être utiles en tant que composition pharmaceutique, composition alimentaire et composition cosmétique pour la prévention ou le traitement de troubles métaboliques, en particulier, une stéatose hépatique tel qu'une stéatose hépatique non alcoolique, une dyslipidémie telle que l'hyperlipidémie ou l'hypertriglycéridémie, et l'obésité, et le procédé de criblage utilisant le mécanisme peut être utile en tant que procédé de criblage d'un matériau pour la prévention ou le traitement de troubles métaboliques.
PCT/KR2016/002410 2015-08-31 2016-03-10 Composition pharmaceutique pour la prévention ou le traitement de troubles métaboliques, contenant du sphingosine-1-phosphate ou un matériau augmentant l'expression de sphk2 WO2017039096A1 (fr)

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CN108853097A (zh) * 2018-08-23 2018-11-23 天津医科大学代谢病医院 噻唑烷二酮拟似物k145在制备治疗肥胖、非酒精性脂肪肝以及高脂血症药物的用途
CN108853097B (zh) * 2018-08-23 2021-05-14 天津医科大学代谢病医院 噻唑烷二酮拟似物k145在制备治疗肥胖、非酒精性脂肪肝以及高脂血症药物的用途

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