WO2008069517A1 - Composition and method for inhibition of lipogenesis by suppressing the expression of protein kinase c delta binding protein - Google Patents

Composition and method for inhibition of lipogenesis by suppressing the expression of protein kinase c delta binding protein Download PDF

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WO2008069517A1
WO2008069517A1 PCT/KR2007/006187 KR2007006187W WO2008069517A1 WO 2008069517 A1 WO2008069517 A1 WO 2008069517A1 KR 2007006187 W KR2007006187 W KR 2007006187W WO 2008069517 A1 WO2008069517 A1 WO 2008069517A1
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pkcδbp
expression
sirna
inhibition
composition
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French (fr)
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Pil-Joon Park
Min-Jeong Song
Si-Young Cho
Tae-Ryong Lee
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Amorepacific Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present invention relates to a composition for the inhibition of adipocytic differentiation, comprising a regulator for downregulating the expression of protein kinase C delta binding protein (PKC ⁇ bp) as an active ingredient, and a method of inhibiting adipocytic differentiation by downregulating the expression of PKC ⁇ bp.
  • PKC ⁇ bp protein kinase C delta binding protein
  • a lipid droplet is a subcellular organelle composed of phospholipids, such as triacylglycerol (TAG), diacylglycerol and cholesteryl ester, and phospholipid- specific proteins responsible for storage or transport of the phospholipids.
  • TAG triacylglycerol
  • cholesteryl ester phospholipid-specific proteins responsible for storage or transport of the phospholipids.
  • diseases associated with the formation of the lipid droplet have been issued. For example, abnormally enlarged lipid droplets may cause diseases such as fatty liver, obesity, arteriosclerosis and diabetes. In particular, with respect to obesity, the size of lipid droplets is increased by two-fold or more, thereby increasing the accumulation of neutral lipids.
  • phospholipid-specific proteins constituting lipid droplets are expressed in an adipocyte-specif ⁇ c manner, and play an important role in carrying TAG to lipid droplets or protecting lipid droplets from lipolysis by lipases. Therefore, in order to prevent or treat lipid droplet-associated diseases such as obesity, there have been numerous attempts to develop a method for regulating lipolysis or lipid accumulation by modulating the expression or activity of phospholipid-specific proteins (Murphy et al., Prog. Lipid Res., 40, 325-438, 2001). However, the essential mechanism of the formation of the lipid droplet or its regulation still remains unresolved.
  • PKC ⁇ bp Protein kinase C delta binding protein
  • the present inventors have therefore endeavored to develop a candidate substance for effectively inhibiting adipocytic differentiation, and have found that PKC ⁇ bp is increasingly expressed during adipocytic differentiation, the expression of 54 kDa PKC ⁇ bp is increased in white adipose tissues, and the downregulation of PKC ⁇ bp expression reduces adipocytic differentiation and intracellular neutral lipid content.
  • a composition for the inhibition of adipocytic differentiation comprising a regulator for downregulating the expression of protein kinase C delta binding protein (PKC ⁇ bp) as an active ingredient.
  • PKC ⁇ bp protein kinase C delta binding protein
  • Fig. 1 Western blot analysis for PKC ⁇ bp expression levels in cells harvested 2, 5 and 8 days after induction of the differentiation of preadipocytes;
  • Fig. 2 Inhibition of PKC ⁇ bp expression by siRNA treatment, determined by a real-time PCR (RT-PCR);
  • Fig. 3a Intracellular neutral lipid contents measured 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp-specific siRNA (PKC ⁇ bp siRNA)-expressing lentivirus, respectively;
  • Fig. 3b Microscopic cell images observed 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp siRNA-expressing lentivirus, respectively;
  • Fig. 4 Graphs showing changes in expression levels of adipogenesis- associated marker genes after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp siRNA-expressing lentivirus, respectively;
  • Fig. 5 Western blot analysis for adipogenesis-associated marker proteins 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp siRNA-expressing lentivirus, respectively;
  • Fig. 6 RT-PCR result showing the expression level of PKC ⁇ bp in preadipocytes transfected with PKC ⁇ bp overexpression lentivirus
  • Fig. 7 Intracellular neutral lipid contents measured 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp overexpression lentivirus, respectively;
  • Fig. 8 Graphs showing changes in expression levels of adipogenesis- associated marker genes after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKC ⁇ bp overexpression lentivirus, respectively;
  • Fig. 9 RT-PCR result showing the expression level of PKC ⁇ bp in tissues harvested after intraperitoneal injection of PKC ⁇ bp siRNA into mice.
  • Mouse protein kinase C delta binding protein (PKC ⁇ bp) is present in adipocytes and other tissues. 54 kDa PKC ⁇ bp is preferentially expressed in white adipose tissues and is increasingly expressed during adipocytic differentiation
  • the present invention provides a composition for the inhibition of adipocytic differentiation comprising a regulator for downregulating PKC ⁇ bp expression as an active ingredient.
  • the regulator used in the inventive composition may be antisense RNAs, interfering RNAs (iRNAs), small interfering RNAs (siRNAs), and expression vectors thereof capable of introducing these RNAs into cells, which can be used for knock-down of the expression of PKC ⁇ bp at its mRNA level; transcription inhibitors of PKC ⁇ bp gene; translation inhibitors of transcribed PKC ⁇ bp mRNA; or inhibitors of PKC ⁇ bp localization.
  • PKC ⁇ bp siRNA and its expression vector are preferable because they can specifically and potently downregulate the expression of PKC ⁇ bp gene even when a small amount is applied thereto.
  • the regulator is a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 1 and 2 or a pair of nucleotide sequences as set forth in SEQ ID NOS: 3 and 4, or its expression vector.
  • composition of the present invention may further comprise pharmaceutically acceptable carriers, excipients or other additives.
  • inventive composition is in the form of an injection formulation for gene therapy.
  • the inventive composition may be systemically or topically administered to a subject in need of inhibition of adipocytic differentiation.
  • a suitable single dose of the active ingredient of the inventive composition may be about from 0.001 ng to 1000 ⁇ g, so that PKC ⁇ bp expression can be downregulated by 70% or more.
  • the dose should be determined in light of various relevant factors including the condition to be treated, the route of administration, the age and weight of a patient, and the severity of the patient's symptoms.
  • the present invention also provides a method of inhibiting adipocytic differentiation by downregulating the expression of PKC ⁇ bp in a subject in need of the inhibition of adipocytic differentiation.
  • the subject may be any mammal such as a human.
  • the downregulation of PKC ⁇ bp expression may be performed by knock-down of the expression of PKC ⁇ bp gene; knock-down of the expression of PKC ⁇ bp mRNA using antisense RNA, RNAi, siRNA, or its expression vector which can introduce one of these RNAs into cells; suppressing the transcription of PKC ⁇ bp gene; suppressing the translation of transcribed PKC ⁇ bp mRNA; or inhibiting the intracellular localization of PKC ⁇ bp.
  • the methods mentioned above should not be construed to limit the scope of the invention. According to the inventive inhibition method, it is possible to effectively inhibit adipocytic differentiation. (J. T.
  • the inventive inhibition method may be beneficially used for preventing or treating diseases associated with a disorder of lipid droplets, including fatty liver, obesity, arteriosclerosis and diabetes (James M. Ntambi et al, PNAS, 99: 11482-11486, 2002).
  • the present invention also provides a method for screening a candidate substance inhibiting adipocyte differentiation using PKC ⁇ bp gene or an antibody specific to PKC ⁇ bp.
  • the inventive screening method may comprise treating cell lines (e.g., differentiated adipocytic cell lines), tissues or test animals, which express PKC ⁇ bp, with candidate substances, and analyzing the changes in expression of PKC ⁇ bp gene or anti-PKC ⁇ bp antibody.
  • the expression level of PKC ⁇ bp gene or protein may be analyzed using one of known methods used for detecting gene or protein expression level.
  • PKC ⁇ bp gene expression may be analyzed by reverse-transcription PCR, or blot analysis such as Northern blot using PKC ⁇ bp gene or a fragment thereof as a probe
  • PKC ⁇ bp protein expression may be analyzed by way of conducting enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blot, immunoblot or immunohistochemical staining, using anti- PKC ⁇ bp antibodies.
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • sandwich assay Western blot
  • Western blot immunoblot
  • immunoblot immunohistochemical staining
  • PKC ⁇ bp expression is closely associated with intracellular lipogenesis. Consequently, the downregulation of PKC ⁇ bp expression can lead to effective inhibition of adipocytic differentiation, and thus significant reduction of neutral lipids. Therefore, it is possible to treat or prevent diseases associated with a disorder of lipid droplets, including fatty liver, obesity, arteriosclerosis and diabetes. It is also possible to screen candidate inhibitors for adipocytic differentiation using PKC ⁇ bp gene or anti- PKC ⁇ bp antibodies.
  • Example 1 The expression level of PKC ⁇ bp depending on adipocytic differentiation
  • 3T3-L1 cells were cultured successively in: DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-l-methylxanthine (Sigma), 1 ⁇ M dexamethasone (Sigma) and 167 nM insulin (Novo-Nordisk) under the condition of 37 °C and 10% CO 2 for 48 hours; DMEM supplemented with 10% FBS and 167 nM insulin under the same conditions for 48 hours; and DMEM supplemented with 10% FBS under the same conditions for 48 hours, to obtain differentiated adipocytes.
  • DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-l-methylxanthine (Sigma), 1 ⁇ M dexamethasone (Sigma) and 167 nM insulin (Novo-Nordisk) under the condition of 37 °C and 10% CO 2 for 48 hours
  • DMEM supplemented with 10% FBS and 167 nM insulin under the same conditions for 48
  • Cells of mouse preadipocytic cell lines, 3T3-L1 were subjected to differentiation for 8 days as described in step 1. Aliquots of the cells were harvested 2, 5 and 8 days from the induction of the differentiation. Each harvested cell sample was treated with 1 ml of RIPA buffer (IxPBS, 1 % Nonidet P 40, 0.5 % sodium deoxylate and 0.1% SDS), incubated on ice for 30 minutes, and centrifuged at 15,000xg, 4 ° C for 10 minutes to obtain a supernatant. The protein content of the supernatant was determined by a protein dye reagent (Bio-RAD).
  • RIPA buffer IxPBS, 1 % Nonidet P 40, 0.5 % sodium deoxylate and 0.1% SDS
  • Example 2 Construction of lentivirus expressing PKC ⁇ bp siRNA and establishment of cell lines transfected with the virus
  • siRNA duplexes capable of mediating RNA interference (RNAi) against PKC ⁇ bp gene expression i.e., a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 1 and 2 (siRNA- 1) and a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 3 and 4 (siRNA-2), were designed using a siRNA design program (Ambion).
  • SEQ ID NO: 1 5' -GGAGCUUUC AGCCUAAUUUtt-3' (Sense)
  • SEQ ID NO: 2 5' -AAAUUAGGCUGAAAGCUCCtc-3 '
  • Antisense SEQ ID NO: 3: 5' -CCCGUGCUUCAAAUAGAGAttt-3' (Sense)
  • SEQ ID NO : 4 5 ' -UCUCUAUUUGAAGCACGGGtt-3 ' (Antisense)
  • the siRNA- 1 and the siRNA-2 were transfected into previously subcultured 3T3-L1 cells (1x10 5 cells/mil) as follows.
  • 5 ⁇ i of NeoFX (Invitrogen Co.) and 100 ⁇ i of OPTI MEM (Invitrogen Co.) were mixed and the mixture was incubated at room temperature for 10 minutes.
  • 100 ⁇ i of the resultant solution was mixed with 3.75 ⁇ i of each (20 ⁇ M) of siRNA- 1 and siRNA-2, and the mixture was then incubated at room temperature for 10 minutes.
  • the resultant solution was loaded in each well of a 6-well plate, and 2.3 ml of 3T3-L1 cells (IxIO 5 cellsM) was seeded thereto.
  • cloning was performed according to a method disclosed in Maniatis et al. (1982). The insertion of siRNA in the cloned vector was confirmed by DNA sequencing. Then, a RNA fragment extracted from the vector was cloned into lentivirus. The cloning was performed by Macrogen Co.
  • Example 2 The cell lines obtained in Example 2 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation.
  • the cells were harvested 2, 5 and 8 days from the induction of the differentiation, fixed with 3.7% formaldehyde, washed three times with PBS, incubated in a 0.2% Oil red solution for 30 minutes, and washed four times (15 minutes each) with PBS.
  • the stained cells were examined with the naked eye and a Zeiss Axioplan-2 microscope, and the results are shown in FIGS. 3 A and 3B.
  • neutral lipids are stained with red color.
  • Example 4 Analysis for changes in expression levels of adipogenesis- associated marker genes
  • Example 2 The cell lines obtained in Example 2 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation.
  • the cells were harvested 2, 5 and 8 days from the induction of the differentiation, washed twice with PBS, and lysed using 1 ml of a Trizol reagent (Invitrogen Co. USA). Genomic DNA was cleaved with a 1 ml syringe, and incubated at room temperature for 2 minutes. 200 ⁇ i of chloroform was added thereto, and the reaction solution was incubated at room temperature for 5 minutes and ultra-centrifuged at 12,000 rpm for 15 minutes. A supernatant was transferred to a new tube, and 500 ⁇ Jt of isopropyl alcohol was added thereto. The reaction solution was ultra-centrifuged at 12,000 rpm for 10 minutes to precipitate RNA.
  • a Trizol reagent Invitrogen Co. USA
  • cDNA was diluted with distilled water (1 :10), and real-time PCR was performed for 20 ⁇ l of a PCR solution containing ⁇ Ji of cDNA, 10 ⁇ l of Master mix (Qiagen), 1 ⁇ i of assay on demand (Applied Biosystems) for each of PREF-I, PPAR ⁇ , and FAS, and water, using Rotor gene 3000 (Corbett Co.).
  • the results are shown in FIG. 4.
  • FIG. 4 in the cell lines transfected with PKC ⁇ bp siRNA-containing lentivirus, the expression of PREF- 1, which is expressed in preadipocytes, was continuously increased.
  • PPAR ⁇ which is increasingly expressed as differentiation proceeds
  • FAS which is associated with synthesis of neutral lipids
  • cellular proteins i.e., PKC ⁇ bp, PPAR ⁇ , stearoyl CoA dehydrogenase (SCD-I), and ⁇ -tubulin were isolated and quantified by the same procedure as in step 2 of Example 1.
  • Western blot analyses were performed using anti-PKC ⁇ bp (LabFrontier), anti-PPAR ⁇ (Santa cruz Co.), and anti-SCD-1 (Alpha diagnostic Co.) as primary antibodies and anti- ⁇ -tubulin (sigma Co.) as a control antibody by the same procedure as in step 2 of Example 1.
  • Example 5 Construction of PKC ⁇ bp overexpression lentivirus and establishment of cell lines transfected with the virus
  • SEQ ID NO: 6 5' -GGGTACCGGCTGCGCTCTCTAT-S 1 (Reverse) That is, PCR was performed using cDNA of 3T3-L1 adipocytes as a template and a pair of the primers of SEQ ID NOS: 5 and 6 to obtain the nucleotide sequence of PKC ⁇ bp gene.
  • the nucleotide sequence of PKC ⁇ bp gene was inserted into pcDNA3. l/myc-His(-)A vector (Invitrogen Co.).
  • the insertion of PKC ⁇ bp gene into the vector obtained in step 1 was confirmed by DNA sequencing.
  • the nucleotide sequence of PKC ⁇ bp gene extracted from the vector was cloned into lentivirus. The cloning was performed by Macrogen Co.
  • Example 6 Analysis for adipogenesis inhibition and expression levels of adipogenesis-associated marker genes in PKC ⁇ bp overexpression cell lines
  • Example 5 The cell lines obtained in Example 5 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation. The inhibition of adipocytic differentiation was analyzed by the same procedure as in Example 3.
  • Example 5 The cell lines obtained in Example 5 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation. After the induction of differentiation, the expression levels of adipogenesis-associated marker genes in the cells were analyzed by the same procedure as in Example 4.
  • FIG. 8 As shown in FIG. 8, in the test cell lines transfected with PKC ⁇ bp overexpression lentivirus, the expression level of PREF-I, which is expressed in the preadipocytes, was slightly reduced. PPAR ⁇ , which is increasingly expressed as adipocytic differentiation proceeds, and SCD-I and FAS, which are associated with synthesis of neutral lipids, were significantly upregulated, as compared with the control cell lines.
  • Example 7 Analysis for an inhibitory effect of PKC ⁇ bp siRNA on PKC ⁇ bp expression in mice (in vivo test)
  • siRNA duplex having nucleotide sequences of SEQ ID NOS: 7 and 8 was designed based on the siRNA sequences of SEQ ID NOS: 1 through 4.
  • a siRNA polymer was prepared using the siRNA duplex by Integrated DNA Technologies (IDT) Co.
  • mice were killed by separation of the cervical vertebrae.
  • liver tissues and epididymal adipose tissues were isolated from the mice.
  • RNA extraction, cDNA synthesis, and real-time PCR were performed by the same procedure as in Example 4 to analyze the expression level of PKC ⁇ bp gene.

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Abstract

There are provided a composition for the inhibition of adipocytic differentiation, including a regulator for downregulating the expression of protein kinase C delta binding protein (PKCδbp) as an active ingredient, and a method of inhibiting adipocytic differentiation by downregulating the expression of PKCδbp. By employing the composition and method of the present invention, it is possible to significantly reduce an amount of neutral lipids, thereby resulting in treatment or prevention of diseases associated with a disorder of lipid droplets, including fatty liver, obesity, arteriosclerosis and diabetes.

Description

COMPOSITION AND METHOD FOR INHIBITION OF LIPOGENESIS BY SUPPRESSING THE EXPRESSION OF PROTEIN KINASE C DELTA BINDING PROTEIN
Field of the Invention
The present invention relates to a composition for the inhibition of adipocytic differentiation, comprising a regulator for downregulating the expression of protein kinase C delta binding protein (PKCδbp) as an active ingredient, and a method of inhibiting adipocytic differentiation by downregulating the expression of PKCδbp.
Background of the Invention
Most organisms convert surplus nutrient that remains after metabolism into neutral lipids and store the neutral lipids in lipid droplets of adipocytes. A lipid droplet is a subcellular organelle composed of phospholipids, such as triacylglycerol (TAG), diacylglycerol and cholesteryl ester, and phospholipid- specific proteins responsible for storage or transport of the phospholipids. Recently, diseases associated with the formation of the lipid droplet have been issued. For example, abnormally enlarged lipid droplets may cause diseases such as fatty liver, obesity, arteriosclerosis and diabetes. In particular, with respect to obesity, the size of lipid droplets is increased by two-fold or more, thereby increasing the accumulation of neutral lipids.
Most of phospholipid-specific proteins constituting lipid droplets are expressed in an adipocyte-specifϊc manner, and play an important role in carrying TAG to lipid droplets or protecting lipid droplets from lipolysis by lipases. Therefore, in order to prevent or treat lipid droplet-associated diseases such as obesity, there have been numerous attempts to develop a method for regulating lipolysis or lipid accumulation by modulating the expression or activity of phospholipid-specific proteins (Murphy et al., Prog. Lipid Res., 40, 325-438, 2001). However, the essential mechanism of the formation of the lipid droplet or its regulation still remains unresolved.
Protein kinase C delta binding protein (PKCδbp), which was first found in humans and mice, is a 37 kDa or 54 kDa protein (Yasushi izumi et al., JBC, Vol. 22, No.l l, 7381-7389, 1997). However, specific functions of PKCδbp have not yet been identified.
The present inventors have therefore endeavored to develop a candidate substance for effectively inhibiting adipocytic differentiation, and have found that PKCδbp is increasingly expressed during adipocytic differentiation, the expression of 54 kDa PKCδbp is increased in white adipose tissues, and the downregulation of PKCδbp expression reduces adipocytic differentiation and intracellular neutral lipid content.
Summary of the Invention
It is an object of the present invention to provide a composition for effectively inhibiting adipocytic differentiation.
It is another object of the present invention to provide a method of effectively inhibiting adipocytic differentiation.
In accordance with one aspect of the present invention, there is provided a composition for the inhibition of adipocytic differentiation, comprising a regulator for downregulating the expression of protein kinase C delta binding protein (PKCδbp) as an active ingredient. In accordance with another aspect of the present invention, there is provided a method of inhibiting adipocytic differentiation by downregulating the expression of PKCδbp in a subject in need of the inhibition of adipocytic differentiation. Brief Description of the Drawings
The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:
Fig. 1 : Western blot analysis for PKCδbp expression levels in cells harvested 2, 5 and 8 days after induction of the differentiation of preadipocytes;
Fig. 2: Inhibition of PKCδbp expression by siRNA treatment, determined by a real-time PCR (RT-PCR);
Fig. 3a: Intracellular neutral lipid contents measured 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp-specific siRNA (PKCδbp siRNA)-expressing lentivirus, respectively; Fig. 3b: Microscopic cell images observed 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp siRNA-expressing lentivirus, respectively;
Fig. 4: Graphs showing changes in expression levels of adipogenesis- associated marker genes after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp siRNA-expressing lentivirus, respectively;
Fig. 5: Western blot analysis for adipogenesis-associated marker proteins 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp siRNA-expressing lentivirus, respectively;
Fig. 6: RT-PCR result showing the expression level of PKCδbp in preadipocytes transfected with PKCδbp overexpression lentivirus;
Fig. 7: Intracellular neutral lipid contents measured 2, 5 and 8 days after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp overexpression lentivirus, respectively;
Fig. 8: Graphs showing changes in expression levels of adipogenesis- associated marker genes after induction of the differentiation of preadipocytes transfected with scrambled lentivirus and PKCδbp overexpression lentivirus, respectively; and
Fig. 9: RT-PCR result showing the expression level of PKCδbp in tissues harvested after intraperitoneal injection of PKCδbp siRNA into mice.
Detailed Description of the Invention
Mouse protein kinase C delta binding protein (PKCδbp) is present in adipocytes and other tissues. 54 kDa PKCδbp is preferentially expressed in white adipose tissues and is increasingly expressed during adipocytic differentiation
The present invention provides a composition for the inhibition of adipocytic differentiation comprising a regulator for downregulating PKCδbp expression as an active ingredient. The regulator used in the inventive composition may be antisense RNAs, interfering RNAs (iRNAs), small interfering RNAs (siRNAs), and expression vectors thereof capable of introducing these RNAs into cells, which can be used for knock-down of the expression of PKCδbp at its mRNA level; transcription inhibitors of PKCδbp gene; translation inhibitors of transcribed PKCδbp mRNA; or inhibitors of PKCδbp localization. Among them, PKCδbp siRNA and its expression vector are preferable because they can specifically and potently downregulate the expression of PKCδbp gene even when a small amount is applied thereto. More preferably, the regulator is a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 1 and 2 or a pair of nucleotide sequences as set forth in SEQ ID NOS: 3 and 4, or its expression vector.
The composition of the present invention may further comprise pharmaceutically acceptable carriers, excipients or other additives. Preferably, the inventive composition is in the form of an injection formulation for gene therapy.
The inventive composition may be systemically or topically administered to a subject in need of inhibition of adipocytic differentiation. A suitable single dose of the active ingredient of the inventive composition may be about from 0.001 ng to 1000 μg, so that PKCδbp expression can be downregulated by 70% or more. However, it should be understood that the dose should be determined in light of various relevant factors including the condition to be treated, the route of administration, the age and weight of a patient, and the severity of the patient's symptoms.
The present invention also provides a method of inhibiting adipocytic differentiation by downregulating the expression of PKCδbp in a subject in need of the inhibition of adipocytic differentiation. The subject may be any mammal such as a human.
In the inhibition method of the present invention, the downregulation of PKCδbp expression may be performed by knock-down of the expression of PKCδbp gene; knock-down of the expression of PKCδbp mRNA using antisense RNA, RNAi, siRNA, or its expression vector which can introduce one of these RNAs into cells; suppressing the transcription of PKCδbp gene; suppressing the translation of transcribed PKCδbp mRNA; or inhibiting the intracellular localization of PKCδbp. The methods mentioned above should not be construed to limit the scope of the invention. According to the inventive inhibition method, it is possible to effectively inhibit adipocytic differentiation. (J. T. Tansey et al., PNAS, 98: 6494-6499, 2001). It is also possible to effectively reduce the intracellular content of neutral lipids, the major targets for curing obesity. Therefore, the inventive inhibition method may be beneficially used for preventing or treating diseases associated with a disorder of lipid droplets, including fatty liver, obesity, arteriosclerosis and diabetes (James M. Ntambi et al, PNAS, 99: 11482-11486, 2002).
The present invention also provides a method for screening a candidate substance inhibiting adipocyte differentiation using PKCδbp gene or an antibody specific to PKCδbp. The inventive screening method may comprise treating cell lines (e.g., differentiated adipocytic cell lines), tissues or test animals, which express PKCδbp, with candidate substances, and analyzing the changes in expression of PKCδbp gene or anti-PKCδbp antibody.
In the inventive screening method, the expression level of PKCδbp gene or protein may be analyzed using one of known methods used for detecting gene or protein expression level. For example, PKCδbp gene expression may be analyzed by reverse-transcription PCR, or blot analysis such as Northern blot using PKCδbp gene or a fragment thereof as a probe, and PKCδbp protein expression may be analyzed by way of conducting enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blot, immunoblot or immunohistochemical staining, using anti- PKCδbp antibodies.
As described above, PKCδbp expression is closely associated with intracellular lipogenesis. Consequently, the downregulation of PKCδbp expression can lead to effective inhibition of adipocytic differentiation, and thus significant reduction of neutral lipids. Therefore, it is possible to treat or prevent diseases associated with a disorder of lipid droplets, including fatty liver, obesity, arteriosclerosis and diabetes. It is also possible to screen candidate inhibitors for adipocytic differentiation using PKCδbp gene or anti- PKCδbp antibodies.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Example 1: The expression level of PKCδbp depending on adipocytic differentiation
<Step 1> Culture and induction of differentiation of adipocytes
Cells of mouse undifferentiated adipocytic cell lines, 3T3-L1 (ATCC
No. CL-173) were cultured in Dulbecco's modified Eagle's medium (DMEM,
Gibco CA. 1210-0038) supplemented with 10% bovine serum under the condition of 37°C and 10% CO2 with medium replacement every 48 hours until
95% confluency. Then, 3T3-L1 cells were cultured successively in: DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-l-methylxanthine (Sigma), 1 μM dexamethasone (Sigma) and 167 nM insulin (Novo-Nordisk) under the condition of 37 °C and 10% CO2 for 48 hours; DMEM supplemented with 10% FBS and 167 nM insulin under the same conditions for 48 hours; and DMEM supplemented with 10% FBS under the same conditions for 48 hours, to obtain differentiated adipocytes.
<Step 2> Analysis for the expression level of PKCδbp
Cells of mouse preadipocytic cell lines, 3T3-L1 (ATCC No. CL-173) were subjected to differentiation for 8 days as described in step 1. Aliquots of the cells were harvested 2, 5 and 8 days from the induction of the differentiation. Each harvested cell sample was treated with 1 ml of RIPA buffer (IxPBS, 1 % Nonidet P 40, 0.5 % sodium deoxylate and 0.1% SDS), incubated on ice for 30 minutes, and centrifuged at 15,000xg, 4 °C for 10 minutes to obtain a supernatant. The protein content of the supernatant was determined by a protein dye reagent (Bio-RAD).
40 μg of the protein from the supernatant was loaded on a 8% SDS-
PAGE gel to perform electrophoresis, and transferred to a PVDF membrane
(Bio-Rad) at 50V for 12 hours. The transferred membrane was blocked with 5% skim milk solution for one hour, and incubated with anti-PKCδbp polyclonal antibody (LabFrontier) or anti-flotillin antibody as a primary antibody, and with HRP (horse radish peroxidase)-conjugated anti-rabbit IgG
(Amersham) as a secondary antibody. The expression of PKCδbp was determined by Western blot using an enhanced chemiluminescence kit (ECL kit, Amersham).
The results are shown in FIG. 1. As shown in FIG. 1, the expression of 54 kDa PKCδbp was dramatically increased from 2 days after the induction of the adipocytic differentiation. Example 2: Construction of lentivirus expressing PKCδbp siRNA and establishment of cell lines transfected with the virus
<Step 1> Analysis for inhibitory effect of PKCδbp siRNA on PKCδbp expression
Two siRNA duplexes capable of mediating RNA interference (RNAi) against PKCδbp gene expression, i.e., a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 1 and 2 (siRNA- 1) and a siRNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 3 and 4 (siRNA-2), were designed using a siRNA design program (Ambion).
SEQ ID NO: 1 : 5' -GGAGCUUUC AGCCUAAUUUtt-3' (Sense) SEQ ID NO: 2: 5' -AAAUUAGGCUGAAAGCUCCtc-3 ' (Antisense) SEQ ID NO: 3: 5' -CCCGUGCUUCAAAUAGAGAttt-3' (Sense) SEQ ID NO : 4 : 5 ' -UCUCUAUUUGAAGCACGGGtt-3 ' (Antisense)
The siRNA- 1 and the siRNA-2 were transfected into previously subcultured 3T3-L1 cells (1x105 cells/mil) as follows. In detail, 5 μi of NeoFX (Invitrogen Co.) and 100 μi of OPTI MEM (Invitrogen Co.) were mixed and the mixture was incubated at room temperature for 10 minutes. 100 μi of the resultant solution was mixed with 3.75 μi of each (20 μ M) of siRNA- 1 and siRNA-2, and the mixture was then incubated at room temperature for 10 minutes. The resultant solution was loaded in each well of a 6-well plate, and 2.3 ml of 3T3-L1 cells (IxIO5 cellsM) was seeded thereto. The mixture was sufficiently stirred and the cells were then cultured for 12 hours. Total RNA was extracted from the cells using Trizol reagent (Invitrogen Co.), and cDNA was synthesized from total RNA using an AMV reverse-transcription system (Promega Co.). The expression level of PKCδ bp was measured by real-time PCR (Corbett Co.).
The results are shown in FIG. 2. As shown in FIG. 2, both siRNA- 1 and siRNA-2 exhibited an excellent inhibitory effect on PKCδbp expression. <Step 2> Construction of lentivirus suppressing PKCδbp mRNA expression and establishment of cell lines transfected with the virus
In order to establish PKCδbp-downregulated cell lines that were transfected with a vector carrying PKCδbp siRNA, the cloning was performed according to a method disclosed in Maniatis et al. (1982). The insertion of siRNA in the cloned vector was confirmed by DNA sequencing. Then, a RNA fragment extracted from the vector was cloned into lentivirus. The cloning was performed by Macrogen Co.
1 ml of each of scrambled lentivirus (Macrogen Co.) as a control and PKCδbp siRNA-containing lentivirus was seeded on 3T3-L1 preadipocytes which were previously subcultured in a 6-well plate, and incubated for 24 hours. The next day, the culture medium was replaced with fresh one. After 24 hours, the cells were transferred to 3 μglmi puromycin-containing DMEM and cultured for 10 days. After the culture, colonies of transfected cell lines were selected.
Example 3: Analysis for adipogenesis inhibition
The cell lines obtained in Example 2 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation.
The cells were harvested 2, 5 and 8 days from the induction of the differentiation, fixed with 3.7% formaldehyde, washed three times with PBS, incubated in a 0.2% Oil red solution for 30 minutes, and washed four times (15 minutes each) with PBS. The stained cells were examined with the naked eye and a Zeiss Axioplan-2 microscope, and the results are shown in FIGS. 3 A and 3B. In FIGS. 3 A and 3B, neutral lipids are stained with red color.
As shown in FIGS. 3A and 3B, the content of neutral lipids was significantly reduced in PKCδbp siRNA-containing cell lines, as compared with the control cell lines. Example 4: Analysis for changes in expression levels of adipogenesis- associated marker genes
The cell lines obtained in Example 2 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation.
The cells were harvested 2, 5 and 8 days from the induction of the differentiation, washed twice with PBS, and lysed using 1 ml of a Trizol reagent (Invitrogen Co. USA). Genomic DNA was cleaved with a 1 ml syringe, and incubated at room temperature for 2 minutes. 200 μi of chloroform was added thereto, and the reaction solution was incubated at room temperature for 5 minutes and ultra-centrifuged at 12,000 rpm for 15 minutes. A supernatant was transferred to a new tube, and 500 μJt of isopropyl alcohol was added thereto. The reaction solution was ultra-centrifuged at 12,000 rpm for 10 minutes to precipitate RNA. RNA was washed with 1 ml of a 75% ethanol solution, ultra-centrifuged at 12,000 rpm for 5 minutes to completely remove residual ethanol, and semi-dried. RNA was solubilized in DEPC- treated water, and quantified by performing absorbance measurement and electrophoresis. cDNA was synthesized from 1 βg/μi of the RNA samples using a reverse-transcription system (Promega). cDNA was diluted with distilled water (1 :10), and real-time PCR was performed for 20 μl of a PCR solution containing \μJi of cDNA, 10μl of Master mix (Qiagen), 1 βi of assay on demand (Applied Biosystems) for each of PREF-I, PPARγ, and FAS, and water, using Rotor gene 3000 (Corbett Co.). The results are shown in FIG. 4. As shown in FIG. 4, in the cell lines transfected with PKCδbp siRNA-containing lentivirus, the expression of PREF- 1, which is expressed in preadipocytes, was continuously increased. PPARγ, which is increasingly expressed as differentiation proceeds, and FAS, which is associated with synthesis of neutral lipids, were significantly downregulated. Meanwhile, cellular proteins, i.e., PKCδbp, PPARγ, stearoyl CoA dehydrogenase (SCD-I), and γ -tubulin were isolated and quantified by the same procedure as in step 2 of Example 1. Western blot analyses were performed using anti-PKCδbp (LabFrontier), anti-PPARγ (Santa cruz Co.), and anti-SCD-1 (Alpha diagnostic Co.) as primary antibodies and anti-γ -tubulin (sigma Co.) as a control antibody by the same procedure as in step 2 of Example 1.
The results are shown in FIG. 5. As shown in FIG. 5, in the control cells (lanes 1, 3, 5, 7), as differentiation proceeded, the expression levels of the three proteins, PKCδbp, PPARγ and SCD-I were increased. On the other hand, in the cells transfected with PKCδbp siRNA-containing lentivirus (lanes 2, 4, 6, 8), even after differentiation was induced, the expression of PKCδ bp was downregulated, and the expression levels of PPARγ and SCD-I were not increased.
Example 5: Construction of PKCδbp overexpression lentivirus and establishment of cell lines transfected with the virus
<Step 1> Construction of PKCδbp overexpression vector
In order to obtain the nucleotide sequence of PKCδbp gene, PCR was performed using the following primers. SEQ ID NO: 5 : 5'- GGAATTCC AATGGGGGAGAGC-S1 (Forward)
SEQ ID NO: 6: 5' -GGGTACCGGCTGCGCTCTCTAT-S1 (Reverse) That is, PCR was performed using cDNA of 3T3-L1 adipocytes as a template and a pair of the primers of SEQ ID NOS: 5 and 6 to obtain the nucleotide sequence of PKCδbp gene. The nucleotide sequence of PKCδbp gene was inserted into pcDNA3. l/myc-His(-)A vector (Invitrogen Co.).
<Step 2> Construction of PKCδbp overexpression lentivirus and establishment of cell lines transfected with the virus
The insertion of PKCδbp gene into the vector obtained in step 1 was confirmed by DNA sequencing. The nucleotide sequence of PKCδbp gene extracted from the vector was cloned into lentivirus. The cloning was performed by Macrogen Co.
1 ml of each of scrambled lentivirus (Macrogen Co.) as a control and PKCδbp overexpresion lentivirus as a test group was seeded on undifferentiated 3T3-L1 adipocytes that were previously subcultured in a 6-well plate, and incubated for 24 hours. The next day, the culture medium was replaced with fresh one. After 24 hours, the cells were transferred to 3 μg/ml puromycin- containing DMEM and cultured for 10 days. After the culture, colonies of transfected cell lines were selected.
Example 6: Analysis for adipogenesis inhibition and expression levels of adipogenesis-associated marker genes in PKCδbp overexpression cell lines
<Step 1> Culture of cell lines transfected with scrambled lentivirus and PKCδ bp overexpression lentivirus Cells of the cell lines obtained in Example 5 were cultured in DMEM supplemented with 10% bovine serum and 3 μg/ml of purimycin under the condition of 37 °C and 10% CO2 until 80% confluency.
<Step 2> Analysis for expression level of PKCδbp gene RNA was extracted from the cell lines obtained in step 1 using a Trizol reagent (Invitrogen Co.), and cDNA was synthesized using an AMV reverse- transcription system (Promega Co.). The expression level of PKCδbp gene was measured by real-time PCR (Corbett Co.).
The results are shown in FIG. 6. As shown in FIG. 6, the expression level of PKCδbp was significantly increased in the cell lines transfected with PKCδbp overexpression lentivirus, as compared with the control cell lines.
<Step 3> Analysis for adipogenesis inhibition
The cell lines obtained in Example 5 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation. The inhibition of adipocytic differentiation was analyzed by the same procedure as in Example 3.
The results are shown in FIG. 7. As shown in FIG. 7, the content of neutral lipids was significantly increased in the test cell lines transfected with PKCδbp overexpression lentivirus, as compared with the control cell lines transfected with scrambled lentivirus.
<Step 4> Analysis for expression levels of adipogenesis-associated marker genes
The cell lines obtained in Example 5 were cultured by the same procedure as in Example 1 to induce adipocytic differentiation. After the induction of differentiation, the expression levels of adipogenesis-associated marker genes in the cells were analyzed by the same procedure as in Example 4.
The results are shown in FIG. 8. As shown in FIG. 8, in the test cell lines transfected with PKCδbp overexpression lentivirus, the expression level of PREF-I, which is expressed in the preadipocytes, was slightly reduced. PPARγ, which is increasingly expressed as adipocytic differentiation proceeds, and SCD-I and FAS, which are associated with synthesis of neutral lipids, were significantly upregulated, as compared with the control cell lines.
Example 7: Analysis for an inhibitory effect of PKCδbp siRNA on PKCδbp expression in mice (in vivo test)
<Step 1> Design of siRNA duplex and polymer
For intraperitoneal injection, a siRNA duplex having nucleotide sequences of SEQ ID NOS: 7 and 8 was designed based on the siRNA sequences of SEQ ID NOS: 1 through 4. SEQ ID NO: 7:
5'- rUrCrUrCrUrArUrUrUrUrGrArArGrCrArCrGrGrGTT-31 (Sense) SEQ ID NO: 8: 5'- rCrCrCrGrUrGrCrUrUrCrArArArUrArGrArGrATT-31 (Antisense)
A siRNA polymer was prepared using the siRNA duplex by Integrated DNA Technologies (IDT) Co.
<Step 2> Injection of PKCδbp siRNA into mice
According to the Institutional Animal Care and Use Committee (IACUC), C57BL male mice (10 weeks old) were maintained on a 12 hour light/dark cycle with free access to food. The siRNA polymer obtained in step
1 was solubilized in a Duplex buffer (100 mM potassium acetate, 30 mM
HEPES, pH 7.5). 10 μi of 400 M siRNA polymer was mixed with 200 fd of a TransIT-QR hydrodynamic delivery solution of a TransIT-QR Hydrodynamic Delivery Starter kit (Minis Bio Corporation, WI, USA), and the mixture was intraperitoneally injected into the mice.
The next day, the mice were killed by separation of the cervical vertebrae. The liver tissues and epididymal adipose tissues were isolated from the mice.
<Step 3> Analysis for expression level of PKCδbp in the liver tissues and epididymal adipose tissues
0.1 g of each of the tissues obtained in step 2 was treated with 1 ml of a Trizol solution and homogenized with a homogenizer. RNA extraction, cDNA synthesis, and real-time PCR were performed by the same procedure as in Example 4 to analyze the expression level of PKCδbp gene.
The results are shown in FIG. 9. As shown in FIG. 9, in the test group, the expression levels of PKCδbp gene in the white epididymal adipose tissue and the liver tissue were reduced by about 15% and about 33%, respectively, as compared with the control group.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

What is claimed is:
1. A composition for the inhibition of adipocytic differentiation, comprising a regulator for downregulating the expression of protein kinase C delta binding protein (PKCδbp) as an active ingredient.
2. The composition of claim 1, wherein the regulator is selected from the group consisting of antisense RNAs, interfering RNAs (iRNAs), and small interfering RNAs (siRNAs) mediating RNA interference (RNAi) against PKCδbp gene expression, and expression vectors thereof; transcription inhibitors of PKCδbp gene; translation inhibitors of transcribed PKCδbp mRNA; and inhibitors of PKCδbp localization.
3. The composition of claim 2, wherein the regulator is siRNA mediating RNA interference (RNAi) against PKCδbp gene expression or its expression vector.
4. The composition of claim 3, wherein the siRNA is a RNA duplex having a pair of nucleotide sequences as set forth in SEQ ID NOS: 1 and 2 or a pair of nucleotide sequences as set forth in SEQ ID NOS: 3 and 4.
5. A method of inhibiting adipocytic differentiation by downregulating the expression of PKCδbp in a subject in need of the inhibition of adipocytic differentiation.
6. The method of claim 5, wherein the downregulation of PKCδbp expression is performed by a method selected from the group consisting of knock-down of PKCδbp gene expression; knock-down of PKCδbp mRNA expression using antisense RNA, iRNA, siRNA, or its expression vector; suppression of the transcription of PKCδbp gene; suppression of the translation of transcribed PKCδbp mRNA; and inhibition of the intracellular localization of PKCδbp.
7. The method of claim 5, wherein the subject is a mammal including a human.
PCT/KR2007/006187 2006-12-08 2007-12-03 Composition and method for inhibition of lipogenesis by suppressing the expression of protein kinase c delta binding protein WO2008069517A1 (en)

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