Patent Application: US-19468608-A

Abstract:
the present invention relates to methods for improving the viability , recovery and functionality of islets that are separated from a donor organ for subsequent transplantation and more particularly relates to the use of eif - 5a1 sirnas to enhance the viability and functionality of islets .

Description:
eukaryotic initiation factor 5a ( eif5a ) appears to be a crucial factor in the post - transcriptional regulation of stress - induced genes , and appears to promote cytokine - mediated apoptosis in mammalian cells . eif5a is a small acidic protein that is very highly conserved throughout eukaryotes , and is the only protein known to contain the unique polyamine - derived amino acid hypusine . eif5a has been proposed to play roles in mrna processing , trafficking , and translation . however , its role , if any , in the inflammatory cascade in pancreatic islets has not before been characterized . pancreatic islets are highly vulnerable to glucose and lipid toxicity , oxidative stress , and inflammatory cytokines . to study the role of factors involved in mediating islet to response cellular stress , the present inventors developed a protocol to deplete islets in vivo of specific proteins using small interfering rnas ( sirnas ). in a study , the inventors sought to determine the role of eif5a in islets by depleting them of eif5a protein using rna interference . however , a major challenge in rna interference studies in primary rodent islets is their relatively poor transfection efficiency . whereas previous work has demonstrated the utility of viral - mediated delivery of short hairpin rnas , this technique is time - consuming to generate the appropriate viruses and is subject to viral toxicity . the inventors discovered a novel protocol to deplete mouse islets of selected proteins in vivo by repeated intraperitoneal injections of stabilized small interfering rnas ( sirnas ). this protocol results in remarkable penetration of sirnas within the islet . this technique can be used to successfully deplete islets of any desired / target protein such as the well - characterized pancreatic transcription factor pdx1 . using this technique , the inventors have also successfully depleted islets of eif5a protein , and demonstrated that eif5a contributes to cytokine - mediated islet dysfunction by promoting translation of the mrna encoding inducible nitric oxide synthase ( inos ). the data suggest a model whereby eif5a is necessary in pancreatic islets for stabilization and / or nuclear export of a subset of mrnas ( possibly those involved in cytokine - mediated stress responses ), including inos mrna . the inventors employed in vivo sirna injection to study the role of eukaryotic initiation factor 5a ( eif5a ) in cytokine - mediated stress . eif5a has been characterized in other systems as a crucial regulator in the translation of stress - induced genes . when sirna against eif5a was injected into mice , a 50 - 70 % decrease in eif5a protein levels in isolated islets was observed . accompanying this fall , a 30 - 40 % increase in glucose stimulated insulin secretion and ca2 + mobilization as compared to control treatment was observed . when isolated islets were exposed to a cocktail of proinflammatory cytokines ( il 1β , tnfα , ifnγ ), the relative enhancement of islet function persisted in animals treated with si - eif5a . islet protection following eif5a knockdown was not accompanied by alterations in the expression of genes whose products are involved in glucose responsiveness or insulin transcription ( s1c2a2 , gck , irs1 , nkx6 - 1 , mafa , pdx1 , neurod1 , and setd7 ). see fig1 c . importantly , although the mrna encoding inducible nitric oxide synthase ( inos ) was upregulated more than 40 - fold in both control - and si - eif5a - treated islets in response to cytokines ( see fig1 a ), inos protein levels were 3 - 5 - fold lower in si - eif5a - treated islets ( see fig1 b ). these data suggest that enhanced function in si - eif5a - treated islets may be secondary to reduced nitric oxide production . this provides evidence that eif5a plays an essential role in islet β cell response to stress signals via control of inos translation , and as such , eif5a may serve as a viable target for therapeutic strategies aimed at preserving viability as well as the ability of the islet cells to function after harvesting and post implantation . the inventors have further shown that blockage of eif - 5a function via inhibition of hypusination ( via administration of gc - 7 — an inhibitor of dhs ) effectively reduces inos levels ). see fig1 d . it has been previously shown that sirna incorporation into islets can be achieved by pancreatic perfusion via retrograde portal vein inoculation . see bradley , et al ., transplantation proceedings , 37 , 233 - 236 , 2005 . briefly , cy - 3 labeled luciferase ( luc ) sirna gl2 duplex was used either packaged with lipofectamine 2000 or unpackaged , and injected either through tail vein ( in vivo , 50 μg per mouse ) or directly into the pancreas by retrograde portal vein inoculation ( in situ , 2 μg per mouse ). pancreata were procured and stored at 4 ° c . for 24 hours after in situ delivery , or 4 hours after in vivo delivery , and islets were isolated and cultured an extra 16 hours before examination . to visualize sirna distribution , pancreata were stained for insulin and examined under a fluorescent microscope . isolated islets were directly examined under a fluorescent microscope . unpackaged sirna reached islets to a similar extent as observed using liposomal - packaged sirna , agreeing with reports of so - called “ naked ”- sirna delivery in vivo . lewis et al ., nat . genet . 32 : 107 - 108 , epub 2002 jul . 2029 , 2002 and mccaffrey a p , et al ., nature 418 : 38 - 39 , 2002 ). fig1 shows that perfusion to the islet cells provides a suitable delivery mechanism to the islet cells . accordingly , the present invention provides a method for inhibiting expression of a target protein in islet cells comprising administering sirna targeted to the mrna encoding the target protein to the islet cells , wherein the sirna inhibits expression of the target protein in the islet cells . administration may be through any means , preferably trough intraperitoneal administration to the islet β cell donor before the islet β cell isolation . in one experiment , the inventors have shown that three daily intraperitoneal injections of stabilized , cy3 - labeled double - stranded rnas into c57bl / 6 mice revealed remarkable penetration into isolated islets , as determined by confocal microscopy . to test this technique for protein knockdown , stabilized sirnas targeted against pdx1 were used . two distinct sirnas against pdx1 message resulted in 50 % and 90 % knockdown of pdx1 protein in islets ( as assessed by immunoblot analysis ). see fig1 a - d . the present invention provides a method for inhibiting expression of eif - 5a1 in islet cells comprising administering eif - 5a1 sirna to the islet cells , wherein the eif - 5a1 sirna inhibits expression of eif - 5a1 in the islet cells . fig1 a and 16b show that administration to the islet cell donor via ip injection also provides a suitable delivery mechanism to the islet cells . fig1 b shows that three daily intraperitoneal injections of stabilized , cy3 - labeled double - stranded rnas into c57bl / 6 mice revealed remarkable penetration into isolated islets , as determined by confocal microscopy . fig1 b and 18b show that the eif - 5a1 sirna treated islet cells do indeed express less eif - 5a1 sirna . by inhibiting eif - 5a1 expression , apoptosis is also inhibited . fig4 and 5 show that treating islets cells with eif - 5a1 sirna prior to isolation provided a mechanism to inhibit these cells from apoptosis ( as demonstrated by a reduction of the number of cells in the sub - g1 phase ). accordingly , the present invention also provides a method for inhibiting apoptosis in harvested islet cells comprising administering eif - 5a1 sirna to the islet β cell donor , wherein the eif - 5a1 sirna inhibits expression of eif - 5a1 in the islet cells and wherein the inhibition of eif - 5a1 expression inhibits apoptosis . preferably the islets cells are treated with eif - 5a1 sirna cells prior to the harvesting process . the present invention further provides a method for preserving the functionality of harvested islet cells after isolation comprising administering eif - 5a1 sirna to the islet cells of an islet cell donor prior to islet isolation , which results in a inhibition or reduction of eif - 5a1 expression in the islet cells , which in turn inhibits apoptosis in the islet cells , as well as preserves their functionality . preserving the functionality of the islet cells means that not only do the islet cells survive the harvesting process , but also maintain their function ( the functionality of the cells remains longer and / or they maintain a better glucose response than non eif - 5a1 sirna treated islet cells )( i . e . release insulin in response to glucose ) after the harvesting process . in one experiment , to investigate the consequence of eif - 5a1 knockdown on islet function and overall health , c57bl / 6 male mice were given intraperitoneal ( ip ) injections of either : vehicle ( 0 . 9 % saline ) [ group i ], control sirna ( 16 . 6 mg / kg ) [ group ii ], or eif - 5a1 sirna ( 16 . 6 mg / kg ) [ group iii ]. injections of the assigned treatment were administered each day for three days ( days − 3 , − 2 , − 1 ) prior to islet harvest ( day 0 ). isolated islets were purified by collagenase treatment and allowed to rest for 16 - 18 hours before testing commenced . after establishing a baseline of function on day 1 post - harvest , half of the islets were exposed to a cytokine cocktail ( 5 ng / ml il1β , 10 ng / ml tnf - α , 100 ng / ml inf - γ ) to mimic conditions pancreatic islets encounter in diabetes type 1 . a schematic of the experimental design and allocation of islets is depicted in fig1 a . protein lysates from each group of islets were analyzed by western blot for protein knockdown . fig1 b illustrates the relative knockdown of eif - 5a1 in group iii treated mouse islets , which is quantified in fig1 c . these data illustrate the efficacy of ip injection to produce knockdown expression of eif - 5a1 in islets . evidence of preserved functionality of the harvested islets is demonstrated by calcium oscillation assays . the inventors have determined that intracellular calcium is an indicator of stimulation and stress . for both humans and rodents , a normally functioning islet responds to glucose stimulation with a biphasic pattern of insulin secretion , as shown in fig1 a . as a first approximation , this biphasic secretion pattern closely resembles similar changes in islet intracellular calcium ([ ca 2 + ] i ), which also consists of a first phase peak that drops into a second phase plateau ( fig1 b ). measurements of [ ca 2 + ] i reported as the ratio of emitted light from 340 and 380 nm stimulation , however , provide greater sensitivity to detect additional features of islet function . the [ ca 2 + ] i response to glucose consists of three phases ( see fig1 ) that roughly reflects the processes described by the “ consensus model ” of beta - cell stimulus - secretion coupling . an initial dip in [ ca 2 + ] i occurs following glucose transport into the beta - cell as the endoplasmic reticulum mobilizes and sequesters [ ca 2 + ] i ( phase 0 ). glucose is then metabolized through glycolysis and further metabolized in the tri - carboxylic acid ( tca ) cycle , which results in atp production . when a sufficient increase in atp / adp occurs to close k atp - ion - channels , a sharp rise in [ ca 2 + ] i occurs due to the opening of l - type calcium channels related to first phase insulin secretion . after this initial first phase peak , [ ca 2 + ] i drops to a plateau that is often punctuated by [ ca 2 + ] i oscillations . the plateau height is directly related to glucose concentration and the rate of second phase insulin secretion . overall , this imaging - based [ ca 2 + ] i measurement is thus a good first approximation of dynamic insulin secretion at the level of the individual islet . many factors can negatively impact the normal [ ca 2 + ] i response to glucose stimulation . disruptions in glucose metabolism , mitochondria and atp production , endoplasmic reticulum ( er ), ion channel function , and myriad other problems can affect the dynamics of calcium handling related to glucose stimulation . by frequently monitoring changes in [ ca 2 + ] i , defects in these aspects of activity are more likely to be observed than by standard measures of static insulin secretion . for example , increased basal [ ca 2 + ] i in low glucose and the loss of the phase 0 dip in [ ca 2 + ] i during glucose stimulation can be indicative of er - stress or possibly ion - channel dysfunction . such defects can not be detected easily by measuring static or kinetic insulin secretion , but the imaging techniques described herein can be utilized to address these questions . in both the presence and absence of cytokines , in calcium oscillations and glucose stimulated insulin secretions studies , the eif - 5a1 sirna ( 16 . 6 mg / kg ) treated islets [ group iii ( eif - 5a1 knockdown islets )] showed a greater response to glucose and an extended maintenance of functionality ( implying extended survival ). see fig1 . in accord , these islets are also expected to show a higher production of insulin ( data pending ). preliminary data from images taken after live / dead staining qualitatively support the notion of eif - 5a1 knockdown extending the life of islet cells ( fig1 ). by comparing the number of red islets , the result of ethd - 1 intercalation into the nuclei of dead islet cells , to the number of green islets that are actively cleaving calcein - am into the fluorescent calcein , a qualitative image of islet viability is ascertained ( fig1 ). the present invention further provides a method of inhibiting apoptosis in cells involved in inflammatory diseases in which the disease is associated with increased production of nitric oxide . for example , inflammatory diseases such as rheumatoid arthritis ( ra ) are associated with increased production of nitric oxide ( no ), due to activation of the inducible nitric oxide synthase ( inos ) pathway . studies in animal models have suggested that no plays a causal role in the pathogenesis of joint inflammation and tissue damage , since the severity of arthritis can be reduced by the administration of nos inhibitors . several cell types present within the joint , including synovial fibroblasts , endothelial cells and chondrocytes , can be induced by pro - inflammatory cytokines to produce no in vitro . moreover , localization studies have shown that there is up - regulation of inos expression in synovial lining cells , chondrocytes and blood vessels in joint tissues obtained from patients with ra . the localization of inos expression to the synovial lining layer and cartilage is of interest in the light of other studies that have shown that apoptosis is increased in ra , particularly in the synovial lining layer and cartilage . the mechanisms responsible for apoptosis in the rheumatoid joint remain unclear , although previous workers have shown that expression of fas antigen is increased in ra synoviocytes and that fas antibody can stimulate the apoptotic death of synoviocytes in vitro . others have investigated the hypothesis that activation of the inos pathway is also involved in stimulating apoptosis in the rheumatoid joint , since high levels of no are known to stimulate apoptosis in many cell types in vitro and have concluded that no acts as a mediator of apoptosis in ra . see r . j . van &# 39 ; t hof et al ., rheumatology 200 , 39 : 1004 - 1008 . as such , the present invention provides a method of inhibiting apoptosis in cells associated with ra ( such as synovial and cartilage cells ) by inhibiting expression of eif5a and hence inhibits or decreases the production of nitric oxide via the inos pathway to inhibit apoptosis in the cells . the present invention further provides a method of inhibiting no production in a cell or host by administration to the cell or host an sirna directed against eif - 5a . the sirna decreases expression of eif - 5a and in turn decreases the production of no by inhibiting the activation of the inos pathway . the invention also provide the use of eif5a sirna to manufacture a medicament to decrease production of nitric oxide via the inos pathway . any eif - 5a1 sirna that inhibits expression of eif - 5a1 may be used . the term “ inhibits ” also means reduce as compared to levels that would occur in control cells , i . e . those cells not having been treated with eif - 5a1 sirna . one exemplary and especially preferred eif - 5a1 sirna comprises the sequence : 5 ′- aaaggaaugacuuccagctgadtdt - 3 ′. co - pending application ser . no . 11 / 293 , 391 , which was filed on nov . 28 , 2005 ( which is herein incorporated by reference in its entirety ) provides additional exemplary eif - 5a1 sirnas and other antisense constructs that have been used to inhibit expression of eif - 5a1 in other cell types and were also shown to inhibit apoptosis . one skilled in the art could design other eif - 5a1 sirnas given the eif - 5a1 sequence and can easily test for the sirnas ability to inhibit expression without undue experimentation . fig6 - 11 provide sequences of eif - 5a1 , exemplary eif - 5a1 sirnas and antisense constructs . in another embodiment of the invention , antisense constructs of eif - 5a1 may be used to inhibit expression of eif - 5a1 and thus inhibit apoptosis of the islet cells , as well as maintain or preserve their functionality . in preferred embodiments the eif - 5a1 sirna targets the following nucleotide sequence of eif - 5a1 ( see fig1 ): 5 ′- aaaggaatgacttccagctga - 3 ′; 5 ′- aagatcgtcgagatgtctact - 3 ′; 5 ′- aaggtccatctggttggtatt - 3 ′; and 5 ′- aagctggactcctcctacaca - 3 ′. in especially preferred embodiments , the sirna targets the following sequence of eif - 5a1 : 5 ′- aaaggaatgacttccagctga - 3 ′. the present invention also provides a method for inhibiting islet cells from undergoing apoptosis during a donor harvesting process . as discussed above , many islets cells undergo apoptosis when they are harvested . the present inventors have shown that providing eif - 5a1 sirna to the islet cells prior to harvesting , offers a protective benefit against apoptosis . the eif - 5a1 sirna is administered to the islet cells of an islet cell donor prior to islet isolation . the donor ( and hence islet cells ) may be any animal , including human islet cells . any method of administration may be used . for example , the sirna may be administered via perfusion through the portal vein of the islet cell donor or via hydrodynamic perfusion through the portal vein of the islet cell donor . see example 1 . another form of administration includes intraperotoneal administration . see example 2 and examples 3 - 5 . perfusion through portal vein is similar to canulation of the bile duct , but the needle points the opposite way . the portal vein is exposed by retraction of liver and shifting of visceral organs to the mouse &# 39 ; s left . a preparative knot is made around it and includes the bile duct . after puncturing the vessel a blunted needle is advanced toward the pancreas and the knot is tightened around it . in a mouse model , 1 ml saline or sirna ( 5 μg ) is released slowly , the needle is removed and the knot is closed behind the needle to prevent fluid escape . at this point the mouse is turned around and the bile duct accessed for pancreas digestion . the pancreas may be held longer with sirna . alternatively , it can be removed but kept cold with collagenase longer . regular islet isolation methods are followed and the islets ( 50 ) may be incubated in for 16 hours . the present invention also provides a composition for inhibiting apoptosis in islet cells , comprising eif - 5a1 sirna , wherein the sirna inhibits expression of eif - 5a1 and thereby inhibits apoptosis and maintains the functionality of the islet cells . the composition may comprise other or additional eif - 5a1 sirnas as discussed above . a preferred sirna comprises the nucleotide sequence 5 ′- aaaggaaugacuuccagctgadtdt - 3 ′ mouse islets express eif - 5a . total rna was extracted from isolated mouse islets and rt - pcr was performed for ( β - actin and for eif - 5a1 ( fig1 ). resting non - stimulated islets exhibited positive levels of eif - 5a1 - mrna . eif - 5a1 - mrna levels diminished after e1f5a1 - sirna delivery : portal vein slow perfusion . mice were introduced 1 ml of sirna ( ct ( control ) sequence or eif - 5a1 , 5 μg ) or saline , n = 2 per group , by slow retrograde portal vein perfusion ( fig2 ). pancreata were digested by collagenase irrigation of pancreatic duct and islets were isolated as described by lewis et al ., proc . natl . acad . sci . usa , 102 : 12153 - 12158 epub 12005 aug . 12110 , 2005 . islets ( 50 per mouse ) were incubated for 16 hours . total rna was then extracted and rt - pcr was performed for β - actin and for eif - 5a1 ( fig3 ). ratio of mrna for eif - 5a1 / β - actin was 5 . 24 ( ct - sirna ) and 3 . 01 ( eif - 5a1 - sirna ). fig3 shows that mrna levels of eif - 5a1 were reduced in those cells treated with sirna . this experiment was repeated with n = 3 mice and islets were incubated for rna extraction in triplicates ; results were consistent with initial observation . eif - 5a1 - mrna levels diminished and islet apoptosis rate reduced after e1f5a - sirna delivery : portal vein hydrodynamic perfusion . mice were introduced 1 ml of sirna ( ct or eif - 5a1 , 5 μg ) or saline , n = 2 per group , by hydrodynamic retrograde portal vein perfusion , which was completed within 5 seconds . pancreata were digested by collagenase irrigation of pancreatic duct and islets were isolated . islets were incubated for 16 hours and then divided : one group was stained with propidium iodide for evaluation of apoptosis ( 50 islets per mouse ) and the other group was processed for rt - pcr ( 25 islets per mouse ). levels of mrna for eif - 5a1 / β - actin were again higher in ct - sirna group than in eif - 5a1 - sirna group . apoptosis rate was reduced by 28 . 1 % ( fig4 ). this experiment was repeated with n = 3 , apoptosis rate again diminished ( fig5 ). islets perfusion with biotinylated - sirna . biotinylated - sirna ( 50 μg ) was perfused into islets as described above ( slow perfusion , n = 1 ). pancreas was fixed in formalin for staining . sirna . sirna molecules were synthesized by dharmacon , lafayette , colo . the sequence of the eif - 5a1 and control sirna were : 5 ′ cggaaugacuuccagcugadtdt 3 ′ and 5 ′ agucgaccuucaguaaggcdtdt 3 ′, respectively . rt - pcr . total rna was extracted from cells using qiagen rneasy kit . eif - 5a1 primers : forward 5 ′- gac agt ggg gag gta cga ga - 3 ′; reverse 5 ′- ggg gtg agg aaa acc aaa at - 3 ′. propidium iodide ( pi ) apoptosis stain . single cell suspension of islets was achieved by gentle trypsinization . cells were washed with pbs and added saponin - pi mixture containing 0 . 3 % saponin , edta 1 mm , rnase , 1 % azide , 1 % fcs and 50 μg / ml pi in pbs . cells were thoroughly vortexed and incubated at 4 ° c . in the dark for 6 hours before analyzed for sub - gi population by facs . antibodies and sirna : mouse monoclonal antibody against eif - 5a1 were generated . anti - actin monoclonal antibody ( clone c4 , # 69100 ) was purchased from mp biomedicals . in western blots , near infrared fluorophore labeled secondary antibodies from li - cor were used ( irdye 800 and irdye 700 ). the sirna specific for eif - 5a1 was directed to the following sequence of eif - 5a1 : 5 ′- aaaggaatgacttccagctga - 3 ′. while control sirna was the transcribed sequence , 5 ′- aaagtcgaccttcagtaagga - 3 ′, and was previously shown to not induce knockdown of any known protein . see co - pending u . s . applications ser . no . 11 / 725 , 470 and pct / us07 / 64424 , herein incorporated by reference in their entireties . ip ( intraperotoneal ) injection of mice with sirna : twelve , 8 - to 10 - week old c57bl / 6 male mice were purchased from charles river . male mice were chosen to avoid the metabolic variation that accompanies the female estrous cycle . mice were randomly assigned one of three treatment groups : group i — vehicle treatment ( 0 . 9 % saline ), group ii — control sirna treatment , group iii — eif - 5a1 sirna treatment . each mouse received the select treatment via ip injection with 16 . 6 mg / kg or similar volume ( in the case of group i ). mice were injected each day ( approximately 11 : 00 a . m .) for three days ( days − 3 , − 2 , − 1 ) prior to islet harvest ( day 0 ). islet isolations and cytokine treatment : islet isolations from c57bl / 6 mice were carried out using standard isolation techniques and a protocol approved by the institutional animal care and use committee ( iacuc ). pancreata were stored in hbss supplemented with 4 . 2 mm sodium bicarbonate and 1 % bsa ( invitrogen ) until collagenase digestion . purification was performed by differential centrifugation . whole islets were maintained in phenolred - free dmem solution supplemented with 10 % fbs and 5 % penicillinn / streptomycin . cultures were incubated at 37 ° c . + 5 % co 2 in 5 . 5 mm glucose . purified islets were allowed to recover for 16 - 18 hours before metabolic testing ( calcium oscillations ). cytokine treatment was applied to half of the islets at the end of metabolic testing on day 1 using a prescribed cocktail of physiologically relevant cytokines ( 5 ng / ml il1β , 10 ng / ml tnt - α , 100 ng / ml inf - γ . cytokine treated islets were allowed to rest 24 hours before further testing and protein analysis commenced ( day 2 and day 3 ). immunoblot assays : 10 μg of islet cell extract ( prepared in laemmli buffer containing 200 m dtt and benzonase ) were resolved by electrophoresis on a 4 - 20 % sds - polyacrylamide gel ( invitrogen ) followed by immunoblot analysis using anti - eif - 5a1 mouse monoclonal primary antibody ( at a 1 : 15000 dilution ). immunoblots were imaged and quantitated with the near infra red technology of the odyssey system ( li - cor biosciences ). calcium oscillation assays : pancreatic islets were handpicked from collagenase - digested 8 - to 10 - wk - old c57bl6 mouse pancreas ( as described above ). islets were treated for 20 minutes in fura - 2 low glucose ( 3 mm ) solution then placed in a temperature - controlled flow chamber . low glucose solution ( without fura ) was flowed over the islet to establish a basal calcium measurement ( 5 minutes ). measurements of calcium were obtained using ip lab 4 . 0 software ( bd biosciences ) and are reported as the ratio of emitted light from 340 to 380 nm . after basal calcium levels were established , a high glucose ( 11 mm ) solution was flowed through the chamber ( 15 minutes ) to induce the cellular calcium response of the islets . the calcium response was monitored in 5 - second intervals over the 20 - minute period of observation . islets are disposed of in a 5 % bleach solution upon conclusion of testing . βtc3 cells were transfected with two different pdx - 1 sirna constructs ( constructs a and b ) or controls and subjected to immunoblotting for the proteins pdx - 1 , gapdh and β - actin . see see fig1 a . c57b1 / 6 mice were injected with the same sirnas intraperitoneally once daily for three days , and islets were isolated and purified via collagenase digestion and differential gradient centrifugation . islets were lysed in 2 % sds and subjected to immunoblotting . see fig1 b . isolated islets were loaded with fura2 for 30 minutes before imaging in 3 mm d - glucose . islets were stimulated with 11 mm d - glucose at 300 seconds , and the fura2 ratio was continuously monitored by fluorescence microscopy . see fig1 c . glucose stimulated insulin secretion ( gsis ) was performed using 50 islets from each treatment group . see fig1 d . this example shows that in vivo administration of sirna against eif5a improves islet function ex vivo . fig1 a provides the schematic representation of experimental design . islets were isolated post - treatment and extracts were subjected to immunoblot analysis . see fig1 b . isolated islets were subjected to rt - pcr analysis of genes essential to glucose sensing and insulin transcription ; although gene transcription was inhibited by cytokine treatment ( 4 h incubation with ifng , il1b , tnfa ), no differences between groups were observed . see fig1 c . si - eif5a - treated islets 24 h ( see fig1 d ) and 48 h ( see fig1 f ) after isolation show improved glucose stimulated calcium responses ( gsca ) compared to controls at 24 h ( see fig1 d ) and 48 h ( see fig1 f ) after isolatin . si - eif5a - treated islets demonstrate enhanced gsca in the presence of cytokines 24 h ( see fig1 e ) and 48 h ( see fig1 g ) after isolation . islets isolated from eif5a treated mice showed improved glucose stimulated insulin secretion (“ gsis ”) compared to controls in both the absence ( see fig1 h ) and presence ( see fig1 h ) of 4 h cytokine incubation , as assessed by gsis . cytokine induced of inos protein production is absent in eif5a - deficient cells . islets treated with cytokines were subjected to rt - pcr for inos mrna ; all treatment groups showed dramatic upregulation of inos mrna upon cytokine exposure . see fig1 a . cytokine - 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