Patent Application: US-97347301-A

Abstract:
the eukaryotic mrna 5 ′ cap structure is recognized by eif4e , which plays an essential role in translational control and cell growth . members of a family of proteins called eif4e - binding proteins inhibit the activity of eif4e and consequently repress translation . following exposure of cells to hormones , cytokines and growth factors , 4e - bps become hyperphosphorylated and dissociate from eif4e , to relieve translation inhibition . the phosphorylation events leading to 4e - bp1 dissociation from eif4e are mediated by the p13 - kinase / frap / mtor signaling pathway . the present study addresses the biological importance of 4e - bp1 in vivo by disrupting its gene in the mouse . homozygous 4e - bp1 deficient mice are healthy and develop normally . however , they show an important decrease in white adipose tissue and blood glucose level , and the males show a decrease in total body weight and an increase in resting metabolic rate . primary mouse embryo fibroblasts show accelerated cell growth and enhanced cap - dependent translation , coincident with an increase in eif4e phosphorylation .

Description:
a method of production and the transgenic animals of the present invention is described herein below . in general , these animals are produced by engineering a nucleic acid construct which can disrupt the expression of the endogenous targeted gene ( e . g . 4e - bp1 gene , and more particularly the murine 4e - bp1 gene ). using known methods , this construct is amplified in bacterial cells , purified , and transferred into es cells or isolated oocytes . the transfected es cells can then be injected into blastocysts to generate chimeras . the chimeras which transmit the mutation to their offspring are identified and selected . these animals can then be used as founder animals to obtain different animal lines , derived from breeding with chosen animals . heterozygous animals can then be produced and further mated to generate a hybrid f1 cross . further matings of the f1 heterozygotes produce the wild type , heterozygous and homozygous null mutants of 4e - bp1 ( having both copies of the 4e - bp1 gene disrupted ). the homozygous animals can then serve in a number of experiments . non - limiting examples thereof include : the characterization of their phenotype , and a reconstitution of the 4e - bp1 activity by complementation by a non - endogenous copy of a wild type 4e - bp1 gene or mutant or variant 4e - bp1 gene . an animal ( or cells derived therefrom ) expressing a mutant form of 4e - bp1 gene ( from human , for example ) could be used to screen for compounds which modulate more specifically the mutant form of the 4e - bp1 gene . the present invention therefore strongly indicates that 4e - bp1 is a regulator of fundamental cellular function in vivo . it is thus expected that this cellular function should occur across species . the presence of the 4e - bp1 gene and its conservation among species ( human , mice , rats , fish and lower organisms such as drosophila , support its essential role in physiology . thus , the modulators of 4e - bp1 - eif - 4e interaction identified by the methods and assays of the present invention should find a utility in the treatment of obesity and other metabolic diseases associated with lipid or glucose metabolism malfunction in humans and other animals . the eif4ebp1 gene targeting vector was constructed to replace the splice acceptor site and the first 57 nucleotides of eif4ebp1 exon 2 with the neomycin - resistance gene ( fig1 a ). exon 2 encodes amino acids 47 to 108 , which encompasses the binding domain for eif4e . the disrupted portion of exon 2 encodes amino acids 47 to 66 of 4e - bp1 . following electroporation of linearized targeting vector dna into the j1 - 129 / sv embryonic stem ( es ) cells , 800 g418 resistant colonies were analyzed by southern blotting for homologous recombination . two es clones were found to contain a correct replacement ( fig1 b ). one of these clones , after injection into balb / c blastocysts enabled germ line transmission . heterozygous ( eif4ebp1 +/− ) mice were then crossed to produce homozygous ( eif4ebp1 −/− ) offspring , and the absence of 4e - bp1 expression in these animals was verified by western blotting ( fig1 c ). of note , no compensatory increase in 4e - bp2 or 4e - bp3 protein levels was observed ( data not shown ). the number of eif4ebp1 −/− offspring was consistent with the laws of mendelian inheritance . litters were of normal size , and the mice developed normally . after more than 2 years , no difference of lifespan was observed and the eif4ebp1 −/ − mice show no evidence of illness or tumors according to a gross anatomical analysis . the mice have been followed until death . blood glucose levels , however , were slightly lower in eif4ebp1 −/ − mice (˜ 15 %; table 1 ). this hypoglycemia could not be explained by hyperinsulinemia , as plasma insulin levels were similar in wild - type and knockout mice ( table 1 ). moreover , the amounts and plasma membrane translocation of the glucose transporters glut - 1 and glut - 4 were similar in wild - type and knockout mice ( data not shown ). routine histological examination of the major organs ( e . g . liver and kidneys ) revealed no abnormalities such as , for example , dysplastic tissue . however , a significant ( p & lt ; 0 . 05 ) decrease of approximately 10 % in body weight was observed in homozygous males in comparison to their wild - type littermates ( table 2 ). the difference in weight was not due to hypophagia , as the food intake was the same for both wild - type and eif4ebp1 −/− mice ( data not shown ). the decrease in body weight could be partially accounted for by a striking reduction of ˜ 60 % in white adipose tissue ( wat ) weight in eif4ebp1 −/− males ( table 2 ). this size reduction was specific to adipose tissue , as heart ( table 2 ), and other tissues ( data not shown ) showed no significant weight difference between wild type and eif4ebp1 −/− mice . female eif4ebp1 −/− mice exhibited a similar phenotype ( table 2 ). consistent with the reduced adipose tissue mass , circulating leptin levels were decreased in eif4ebp1 −/− mice (˜ 60 %, table 1 ). triglycerides levels were also measured , but no statistically significant difference was observed between wild - type and knockout mice ( table 1 ). however it appears that there is a sexual dimorphism in this phenotype as the female eif4ebp1 −/− mice do not show the same extent of decrease in their total body weight , even though their fat pads are also decreased in weight (˜ 50 %, table 1 ), as compared to their male counterparts . in view of the reduced adipose tissue in eif4ebp1 −/− mice , it was of interest to determine whether eif4ebp1 disruption might affect glucose homeostasis and metabolic rate . to investigate this possibility , an insulin tolerance test was performed . briefly , the blood glucose concentration of five fed male mice was determined ( 16 : 30 ). five male mice were fasted for 6 h ( 3 : 00 - 9 : 00 am ), and insulin ( eli - lilly ) was injected ( 0 . 4 u / kg ) intraperitoneally . blood was collected serially from retro - orbital sinus or tail vein under anesthesia and blood glucose levels were measured ; the insulin tolerance test was performed twice and the mean and standard deviation from the mean calculated . in the fed state ( fig2 a ), the basal level of glucose after fasting was lower by ˜ 20 % in eif4ebp1 −/− mice ( fig2 b , t = 0 ) as compared to their wild - type littermates . this ratio was maintained following insulin treatment and during recovery ( fig2 b ). thus , the 4e - bp1 −/− mice are not diabetic . this result indicates that the regulation of glucose uptake and metabolism in response to insulin is not altered in eif4ebp1 −/− mice , but rather that some constitutive change in glucose homeostasis has occurred in the eif4ebp1 −/− mice . a glucose tolerance test was also performed , but no significant difference was observed between wild - type and knockout animals ( data not shown ). to further characterize the adipose tissue phenotype , histological sections of white adipose tissue ( wat ) and interscapular brown adipose tissue ( ibat ) were examined . the inguinal and retroperitoneal wat ( iwat and rwat ) of eif4ebp1 −/− mice displayed a striking increase in the number of multilocular adipocytes , which are characteristic of brown adipose tissue ( fig3 a ). furthermore , eif4ebp1 −/− ibat displays smaller lipid droplets ( fig3 a ). these histological observations could be explained if energy expenditure was increased in the knockout mice . consequently , the resting metabolic rate ( rmr ) of eif4ebp1 −/− mice was examined and a significant increase in the males (˜ 15 %; table 3 ) was observed . the difference between the rmr of eif4ebp1 −/− and wild type female mice was not statistically significant ( table 3 ). at the molecular level , a major difference between wat and bat is the expression of an uncoupling protein ( ucp1 ), which uncouples oxidative phosphorylation in the bat inner mitochondrial membrane ( boss et al ., 1998 ). ucp1 is responsible for the increased thermogenesis associated with brown adipocytes ( lowell et al ., 1993 and enerbäck et al ., 1997 ) and its overexpression prevents genetic obesity in mice ( kopecky et al ., 1995 ). the expression levels of ucp1 and ucp2 mrnas in iwat from wild - type and eif4ebp1 −/− mice were thus examined ( fig3 b ). consistent with the increased number of multilocular adipocytes , the mrna expression of ucp1 , but not ucp2 , is increased ˜ 6 fold in iwat ( fig3 c ). thus , the histological , physiological and molecular features of brown adipocytes are all clearly apparent in the white adipose tissue of eif4ebp1 knockout mice . thus , the data herein presented reveal an unanticipated role for 4e - bp1 in the regulation of fat metabolism . the mechanism explaining this regulation is not immediately clear , however . the assessment as to whether the unanticipated role of 4e - bp1 in the regulation of metabolism is through a modulation of protein synthesis was examined in primary embryo fibroblasts ( mefs ) derived from wild type and eif4ebp1 −/− mice . general translation rates were examined by metabolic labelling with 3 h [ leucine ], but no significant change was observed between wild - type and eif4ebp1 −/− mefs ( data not shown ). because 4e - bp1 inhibits cap - dependent , but not cap - independent translation ( pause et al ., 1994 , supra ), the effect of eif4ebp1 disruption on both translation modes was examined . thus , the expression of chloramphenicol acetyltransferase ( cat ) from a construct in which translation is cap - dependent ( t7 - cat ) was compared to that in which translation is directed by an internal ribosome entry site ( ires ) from which translation is cap - independent ( t7 - emcv - cat ) ( fig4 a ). given that elimination of 4e - bp1 should result in increased availability of eif4e for eif4f formation , a preferential enhancement of cap - dependent translation is expected in 4e - bp1 −/− mefs relative to its translation in wild - type mefs . consistent with this prediction , the quantity of cat protein production per rna molecule was 117 % higher in eif4ebp1 −/− mefs than in wild - type mefs ( fig4 b ). in contrast , cat expression from the ires - dependent emcv - cat vector was decreased only slightly ( 15 %; fig4 b ). to ensure that the differences observed in this experiment reflect changes in translation rather than differences in rna levels , an rna quantitation using a real time detection pcr method was performed ( data not shown ), thus yielding a quantity of cat protein per 10 9 rna copies ( fig4 b ). taken together , fig4 a and 4 b show that the elimination of 4e - bp1 resulted in enhancement of cap - dependent translation initiation . since elimination of 4e - bp1 causes an increase in the amount of eif4e that is available for incorporation into eif4f , and that changes in translation caused by eif4e overexpression are associated with changes in cell growth , the growth properties of wild - type and eif4ebp1 −/− primary mouse embryonic fibroblasts ( mefs ) were examined . as shown in fig4 c , eif4ebp1 −/− mefs exhibited faster growth rates ( 10 - 20 %) than wild - type mefs ( fig4 ). this was also evident when cells were kept for longer periods of time in culture ( data not shown ). numerous studies have reported a positive correlation between cell growth and translation rates and eif4e phosphorylation state . morevover , the in vitro phosphorylation of eif4e by two kinases ( mnkl and pkc ) has been shown to be inhibited by its binding to 4e - bp1 . eif4ebp1 −/− mefs were used to study the status of eif4e phosphorylation , which is known to correlate with translation rates and cell growth status ( gingras et al ., 1999 ). eif4e is phosphorylated by the docking of mnk1 , a serine / threonine kinase , on eif4g is prevented by the binding of 4e - bp1 to eif4e ( pyronnet et al ., 1999 ). the deletion of 4e - bp1 in the mouse is thus expected to lead to an increase in eif4e phosphorylation . the effects of 4e - bp1 deletion on eif4e phosphorylation were analyzed by isoelectric focusing ( fig5 b ). indeed , eif4e phosphorylation in mefs was increased from 16 % in wild - type mefs to 44 % in eif4ebp1 −/− cells ( fig5 c ). to confirm that the increase in eif4e phosphorylation in eif4ebp1 −/− cells was caused by the absence of 4e - bp1 , eif4ebp1 −/− mefs were transfected with a 4e - bp1 expression vector ( a knock - in approach ) ( fig5 a ). consistent with the direct role of 4e - bp1 in affecting the phosphorylation status of eif - 4e , the expression of 4e - bp1 in the eif4ebp1 −/− cells led to a decrease (˜ 66 %) in eif4e phosphorylation ( fig5 b and 5 c ). thus , these data provide the evidence that 4e - bp1 can also regulate eif4e phosphorylation . taken together , the data herein presented indicate that 4e - bp1 is a novel mediator of energy homeostasis in mammals . in addition , they identify translation control and more particularly cap - dependent translation as a key process in energy homeostasis in animals . while not being limited to a particular theory , the most likely underlying mechanism to explain these results is the up - regulation of eif4e activity , which would then specifically affect the translation of mrnas involved in brown adipocytes activation and function . one such candidate mrna is the uncoupling protein - 1 ( ucp1 ), a specific marker of brown adipocytes . however , the increase in ucp1 mrna expression cannot be directly linked to the function of eif4e in translation initiation . instead , eif4e might stimulate the translation of an mrna encoding a factor which is involved in brown adipocyte differentiation , mitochondrial biogenesis , or in the up - regulation ucp1 expression . the molecular determinants regulating brown fat cells differentiation are still poorly characterized . one possible candidate is the peroxisome proliferator - activated receptor y ( ppary ), which can specifically transactivate the ucp1 promoter in brown adipocytes ( wu et al ., 1999a ). however , ppary is also expressed in white fat cells and thus cannot explain the specificity of ucp1 induction . another candidate is the ppary coactivator 1 ( pgc1 ), a coactivator of nuclear receptors involved in adaptative thermogenesis and mitochondrial biogenesis ( wu et al ., 1999b ). pgc1 activates ucp1 expression when ectopically expressed in 3t3 - f442a preadipocytes , and ucp2 expression when expressed in c2c12 myotubes . it would thus be interesting to investigate whether the expression of pgc1 mrna is under translational control . finally , it is also possible that eif4e affects the expression of a protein involved in the signaling pathway activating the ucp1 promoter . an eif4ebp1 knockout mouse has been reported previously , but the features of reduced fat tissue and increase multilocular adipocytes in wat described in the present study were not reported or suggested therein , although a reduction in the body weight of male mice was noted ( blackshear et al ., 1997 ). a plausible explanation for this discrepancy might be the different mouse strains used to backcross the f0 mice . while balb / c mice were used herein for crossing to the 129 strain , blackshear et al . used c57bl6 / j . there are numerous reports showing strain - dependent phenotypic changes in mice , especially when assessing metabolic disorders ( see , for example , ewart - toland et al ., 1999 ; surwit et al ., 1995 , coleman et al ., 1973 ; and hummel et al ., 1972 ). moreover , the emergence of brown adipocytes in white fat has been shown to be under a complex genetic control ( guerra et al . 1998 ), which might explain the fluctuations in ucp1 expression levels ( fig3 b ). consequently , the eif4ebp1 −/− mutation is being transferred to the balb / c strain originally used to backcross the 129 chimera . in an inbred background , the eif4ebp1 −/− phenotype should be more readily amenable to metabolic studies . in such a genetic background based on balb / c ( e . g . an inbred background ) variability would be minimized . the implication of cap - dependent translation is glucose metabolism . a large number of studies on cells in culture have provided evidence that eif4e plays an important role in the control of cell growth . the conclusions from the earlier studies are supported by the present results , which provide evidence that translation initiation in animals plays an important role in cell growth and body metabolism . the finding that 4e - bp1 is implicated in the control of fat tissue growth , metabolism , and glucose homeostasis is of pharmacological value , as specific modulation of the 4e - bp1 - eif - 4e interaction , as well as the modulation of the formation of the eif - 4f preinitiation complex and of the level of eif - 4e complex to eif - 4g1 , could be used to modulate fat and glucose metabolism , for example . this possibility is particularly intriguing in light of the fact that 4e - bp1 elimination is not deleterious to mice health . furthermore , the present invention , having identified translation initiation through eif4e and its association with eif - 4g as a biochemical pathway involved in metabolism in vivo , provides numerous assays and methods to screen and identify metabolism modulators and especially fat and glucose metabolism modulators . two functional 4e - bp1 homologs , 4e - bp2 and 4e - bp3 , exist in mammals ( pause et al ., 1994 ; and poulin et al ., 1998 ). although no functional differences have been reported among them , their tissue distribution differs ( poulin et al ., 1998 ; and tsukiyama - kohara et al ., 1996 ). for example , 4e - bp1 is more abundant in wat as compared to the other homologs ( not shown and hu et al ., 1994 ; and lin et al ., 1996 ). it is conceivable that in eif4ebp1 −/− mice , the presence of 4e - bp2 and 4e - bp3 may attenuate a phenotype that would have been observed by the loss of all three proteins . thus , a double , and perhaps a triple knockout , might exhibit a more severe phenotype in wat reduction , and might also show additional phenotypic changes not observed in eif4ebp1 −/− . the actual phenotype of 4e - bp2 , 4e - bp3 single knockout animals or of double or triple knockouts awaits formal testing . as seen in fig6 and 7 , the eif4e binding sites ( or eif4e interaction domains ) of numerous protein from evolutionarily distant organisms show a significant homology / identity . in addition , the sequences of rat and mouse 4e - bp1 , 4e - bp2 and 4e - bp3 are 100 % identical to those of the human in the region presented here . indeed , consensus sequences which retain their eif4e binding activity are provided . for example , a consensus 4e - binding sites of 4e - bps is + φφy −+ xf / aφφxxrxsp wherein + and − refer to a charged amino acid ; φ is a hydrophobic amino acid ; x is any amino acid ; and the capital letters refer to the known one letter code for amino acids . preferably , the consensus sequence has the sequence + φφy −+ xflφxxrxsp , wherein f refers to a preferred but apparently the non - essential amino acid phe ( the rest is as for the previous consensus sequence ). in yet another embodiment , the 4e - binding consensus sequence has the sequence + φxxyx + xfφφ or yxxxxlφ . conversely , they could be used to design eif - 4e or negative regulators thereof , which no longer interact or show lower affinities . these consensus sequences could be used as eif4e sequestering agents or as starting points to design other eif4e sequestering agents . of note , recombinant peptides derived from 4e - bp1 and eif - 4gii have been shown to inhibit translation of mrnas ( marcotrigiano et al ., 1999 , molecul . cell 3 : 707 - 716 ). the present invention is illustrated in further detail by the following non - limiting examples . fragments ( 4 kb ; spe i - sal i ) and ( 3 . 5 kb ; msci - bamhi ) of the murine eif4ebp1 gene were ligated to the 5 ′ and 3 ′ ends of the pgk - neo vector ( polya -) to construct the targeti / ng vector for gene disruption . the dna was digested with spe i and xhol , purified with lgt agarose ( fmc ) and electroporated into es cells ( 129 / sv ) with a gene pulser ( bio - rad ). cells were selected with g418 , as described previously . g418 resistant clones were screened for correct targeting by southern blotting using probe a ( fig1 a , b ), and positives clones were confirmed with probe c ( fig1 a , b ). properly targeted hemizygous es cells were injected into balb / c blastocysts and chimera mice were backcrossed to balb / c mice to generate eif4ebp1 +/− mice . following mating of the heterozygous mice , genomic southern blotting using probe a or b was performed to genotype the progeny mice ( fig1 c ). the absence of 4e - bp1 in mouse tissue was confirmed by northern blot analysis using a portion of exon 2 ( smai - mscl , nt 142 - 203 of the coding region ) as a probe ( data not shown ), and western blotting ( fig1 c ), using anti - 4e - bp1 antibody . mef cells were prepared from 14 day old embryos , as described previously . 4e - bp1 expression vector was constructed as follows : the mouse 4e - bp1 cdna was cloned by rt - pcr using sense primer 5 ′- tgcaggagacatgtcg - 3 ′ and anti - sense primer 5 ′- acagtttgagatggac - 3 ′, with superscriptii ( gibco - brl ) and pfu polymerase ( toyobo ). it was sequenced and subcloned under the control of a cag promoter ( ag promoter with cmv - ie enhancer ). a puromycin resistant cassette was derived from the pbabe - puro vector and introduced into the 4e - bp1 expression vector , which was transfected ( 4 mg ) into mef cells ( 6 cm dishes ) with lipofectin ( gibco - brl ). t7 - cat was described previously and emcv cat was kindly provided by dr . sung - key jang ( pohang institute of science and technology , korea ). one - dimensional vertical slab isoelectric focusing was carried out as described previously using a protean ii minigel apparatus ( bio - rad ). proteins were focused on a 5 % acrylamide gel containing 9 . 5m urea , 3 % ampholine ph4 . 5 - 5 . 5 , 1 % ampholine ph3 . 5 - 10 , 2 % chaps ). histidine ( 10 mm ) was used as the cathode buffer and glutamic acid ( 50 mm ) was used as the anode buffer . focusing was performed for 3 h at 500 - 750 v , followed by transfer of proteins to a polyvinylidene fluoride membrane ( immobilon p , millipore ). filters were probed with anti - eif - 4e rabbit polyclonal antibody as described previously ( frederickson et al ., 1991 ). mef cells were transfected with t7 - cat and t7 - emcv - cat plasmids by lipofectin , followed by infection with a recombinant vaccinia virus expressing the t7 rna polymerase gene ( lot7 - 1 rvv ) as described previously ( takeuchi et al ., 1999 ). rna quantitation was performed by a real time detection pcr method using a sense primer ( 5 ′- gggtgagtttcaccagttttga - 3 ′), an anti - sense primer ( 5 ′- ccactcatcgcagtactgttgt - 3 ′), and a probe ( 5 ′( fam )- caatatggacaacttcttcgcccc -( tamra ) 3 ′), as described previously ( yasui et al ., 1998 ). expressed cat protein was measured by cat - elisa ( roche ). oxygen consumption ( vo 2 ) was simultaneously determined for 4 mice per experiment in an oxymax metabolic chamber ( columbus instruments ). individual mice ( 18 to 24 weeks old ) were placed in a chamber with an airflow of 0 . 5 l / min . ambient temperature was maintained between 24 . 5 and 25 . 5 ° c . experiments for male mice were performed between 12 : 00 pm and 3 : 00 pm , and for females mice between 3 : 00 pm and 6 : 00 pm . mice were placed into the chambers one hour before beginning the experiment to reduce anxiety . five reading were taken at ten minutes intervals over the next 50 minutes and averaged . male animals were either fed ad libitum ( fed ) or fasted for 6 h ( fasted ). serum glucose levels were measured using a one touch basic glucometer ( lifescan canada ltd .). fed insulin levels in serum were measured using a radioimmunoassay ( linco ). fed serum leptin levels were measured by elisa ( r & amp ; d systems ). fed serum triglycerides levels were measured using a triglycerides detection kit ( wako ). these findings show that cap - dependent translation significantly regulates energy homeostasis , and glucose and fat metabolism in animals . more particularly , it identifies the sequestration of eif - 4e as a key determinant in these critical pathways . furthermore , 4e - bp1 is shown to be an important regulator of body metabolism as a consequence of its function as a repressor of translation . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified , without departing from the spirit and nature of the subject invention as defined in the appended claims . 1 . gingras , a . - c ., raught , b . & amp ; sonenberg , n . eif - 4 initiation factors : effectors of mrna recruitment to ribosomes and regulators of translation . annu . rev . biochem . 68 ( 1999 ). 2 . pause , a . et al . insulin - dependent stimulation of protein synthesis by phosphorylation of a regulator of 5 ′- cap function . nature 371 , 762 - 767 ( 1994 ). 3 . poulin , f ., gingras , a . c ., olsen , h ., chevalier , s . & amp ; sonenberg , n . 4e - 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