type 1 diabetes patients often suffer from total loss of their functional  cells , as indicated by the complete absence of insulin c - peptide in their serum .
replacing  cell mass by pancreatic islet transplantation has been shown to be curative in principle but is limited by the availability of donor islets , immunological complications , and transplant survival ( shapiro et  al . , 2006 ) .
therefore , attempts to regenerate patient - specific insulin - producing cells have been undertaken using different cell sources , including embryonic stem cells ( escs ) , induced pluripotent stem cells ( ipscs ) , hepatic cells , exocrine cells , and other endocrine cells ( al - hasani et  al .
, 2009 , kroon et  al . , 2008 , pagliuca et  al . , 2014 , sangan et  al . , 2015 ,
, 2012 , zhou et  al . , 2008 ) . in most cases ,
these approaches have relied on the overexpression of master regulatory transcription factors involved in normal pancreas development , and in only a few cases , small molecules or biologicals have been used ( fomina - yadlin et  al . , 2010 , kubicek et  al . , 2012 , pennarossa et  al . , 2013 , xie et  al . , 2013 , yi et  al . , 2013 ) .
 cells are particularly attractive starting points for transdifferentiation protocols , as they are developmentally closely related to  cells .
these cells have been shown to replenish insulin - producing cells following extreme  cell loss ( thorel et  al . , 2010 , ye et  al . , 2015 ) .
in a genetic model , overexpression of the transcription factor pax4 converts mouse  cells to  cells during development ( collombat et  al . , 2009 ) and when triggered in adulthood ( al - hasani et  al . , 2013 ) .
molecularly , the  cell factor pax4 acts by directly repressing the  cell master regulatory transcription factor arx ( collombat et  al . , 2003 ) , and the loss of arx alone is sufficient to convert  cells into  cells ( courtney et  al . , 2013 ) .
so far , pax4- and arx - mediated  and  cell transdifferentiation has been observed in  vivo in the endogenous environment of a pancreatic islet of langerhans ( collombat et  al . , 2009 , courtney et  al . , 2013 ) .
it remains unclear whether these cell - type conversions occur by a cell - autonomous mechanism or are dependent on other islet cell types and/or signaling from remote organs , such as the liver . to discriminate the two scenarios , we generated cell - line models for inducible overexpression of pancreatic transcription factors and showed their applicability to identify small - molecule modulators of the transdifferentiation process . from a representative library of approved drugs , we identified the anti - malarial drug class of artemisinins as compounds that functionally counteract the  cell - specific transcription factor arx , thereby overcoming a major barrier for  cell transdifferentiation .
we show that these compounds bind to gephyrin , a multifunctional protein that has primarily been studied in neurons ( tyagarajan and fritschy , 2014 ) . among other functions ,
gephyrin is essential for active gabaa receptor signaling , a pathway that has been proposed to reverse diabetes by induction of  cell proliferation ( purwana et  al . , 2014 , soltani et  al . , 2011 ) .
consequently ,  cells acquire  cell characteristics , resulting in improved glucose homeostasis in zebrafish and rodent diabetes models .
to discriminate cell - autonomous effects of transcription factor - mediated transdifferentiation from phenotypes that require paracrine and endocrine signaling in an islet microenvironment , we engineered the mouse  cell line min6 to allow the inducible overexpression of arx .
based on the known heterogeneity of the parental cell line ( nakashima et  al . , 2009 ) , we isolated clonal derivatives with integrations of constructs for the doxycycline - controlled transcriptional activation of myc - tagged arx ( figures s1a and s1b ) . while basal expression levels of  and  cell genes varied in the different subclones , we observed that induction of arx overexpression consistently activated the transcription of  cell genes and repressed  cell - specific transcripts ( figure  s1c ) in a time - dependent manner ( figure  s1d ) .
these changes indicate that our cell lines faithfully model the -to- fate switch upon arx overexpression and suggest that transdifferentiation can be initiated in a cell - autonomous fashion .
furthermore , the cellular system we generated is directly applicable to a high - content screen for functional repressors of arx . in dmso - treated control samples
, we observed a > 50% reduction in insulin protein levels after arx induction compared to uninduced cells ( figures 1a and 1b ) . to identify small molecules that counter arx function
, we induced arx expression and , at the same time , added compounds from a library of 280 clinically approved small molecules selected for their structural and target diversity .
we then measured insulin levels after 72  hr and selected hit compounds for their ability to maintain high insulin levels in the presence of arx while not affecting cell viability ( figure  1c ) .
all compounds that appeared to counteract arx in min6  cells were then tested for their ability to induce insulin expression in the  cell line tc1 ( figure  1d ) .
two artemisinins , artemether and its active metabolite dihydroartemisinin , fully inhibited the arx overexpression phenotype in min6 cells while also inducing insulin expression in tc1 cells , as predicted for functional inhibitors of arx ( figure  1e ) .
importantly , artemether treatment increased insulin expression in min6-arx - induced cells and in tc1 cells in a dose - dependent manner ( figures s2a and s2b ) . to further understand the effects of artemether on  cells
, we analyzed the relative quantitative changes in the proteome of tc1 cells treated with artemether for 72  hr .
proteins with roles in oxidative phosphorylation and mitochondrial localization were significantly altered , with some of these proteins showing increased abundance and others showing decreased abundance . in line with artemisinins acting as functional arx antagonists ,
glucagon protein levels were reduced by more than 50% after artemether treatment ( figure  1f ) .
importantly , artemether reduced the abundance of both proglucagon and processed glucagon peptides in tc1 cells ( figure  1 g ) .
to understand the effect of artemether on arx , we used the min6 overexpression model to study the cellular localization of the transcription factor .
we observed that artemether induced the translocation of arx from the nucleus to the cytoplasm ( figures 1h1j ) , thereby depleting arx from chromatin .
based on the activity of artemether , we investigated the effects of additional artemisinin analogs in  cells .
the closest structural analogs , including arteether and artesunate , showed effects comparable to those of artemether .
in contrast , deoxyarteether , an analog lacking the endoperoxide moiety , did not increase insulin expression in  cells ( figure  s2c ) . to rule out the possibility that artemether , an endoperoxide , induced insulin expression due to the generation of reactive oxygen species ( ros ) , we applied the ros inducer elesclomol .
no significant change of insulin expression in  cells was apparent ( figure  s2d ) . finally , combined treatment with the anti - oxidant
n - acetylcysteine , a known suppressor of the anti - malarial properties of artemisinins ( arreesrisom et  al . , 2007 ) , abolished the effect of artemether on insulin expression in  cells ( figure  s2e ) .
artemisinins are among the most effective therapies for malaria and , based on their rapid toxicity to plasmodia , are the preferred first - line treatment for the disease ( nosten and white , 2007 ) . despite being widely used
different molecular targets have been proposed , including heme binding followed by iron oxidation in human erythrocytes ( shukla et  al . , 1995 ,
wang et  al . , 2015 ) and the inhibition of two different plasmodium - specific proteins : the sarcoplasmic / endoplasmic reticulum calcium atpase ( serca ) pfatp6 ( eckstein - ludwig et  al . , 2003 ) and phosphatidylinositol 3-kinase pfpi3k ( mbengue et  al . , 2015 )
potent effects of artemisinins are also observed on human cells , where these compounds have been assigned anti - inflammatory and anti - cancer properties ( ho et  al . , 2014 ) .
there is limited evidence for possible positive effects of artemisia extracts on animal models of type 1 diabetes ( ahmad et  al . , 2014 ) and in human patients ( al - waili , 1988 ) ,
but the use of isolated artemisinins for the treatment of diabetes has never been proposed , and their effect in the pancreas has not been evaluated . to identify the molecular mechanism of artemisinins in pancreatic  cells , we first assessed whether the homologs of proposed targets of the compounds in plasmodia were involved .
we used the serca inhibitor thapsigargin on tc1 cells and did  not observe increased insulin levels up to dose - limiting toxic  concentrations ( figure  s3a ) .
next , we tested whether artemether treatment of tc1 cells resulted in reduced pi3p and phosphorylated ( p-)akt levels , indicative of pi3k inhibition ( figures s3b and s3c ) . rather than inhibition
, we observed an  increased signal in both readouts , thereby excluding a conserved mechanism of action based on the proposed plasmodium targets . in order to discover potential mechanisms of action of artemisinins in mammalian endocrine cells
, we undertook a chemical proteomics approach ( rix and superti - furga , 2009 ) to determine the proteins that directly interact with artemisinins in tc1 cells .
we coupled artesunate , an artemisinin active in tc1 cells , to solid support and performed pull - down experiments in the presence and absence of competing free artemether ( figure  2a ) .
mass spectrometry identified gephyrin as the most significantly enriched specific interactor ( figures 2b and s3d ) .
artemether increased gephyrin protein levels in tc1 cells ( figures 2c and s3e ) , while mrna expression levels remained unchanged ( figure  s3f ) , suggesting stabilization of the protein . to examine the interaction between gephyrin and artemisinins in a cellular context , we used a fluorescently labeled artelinic acid probe .
the cellular localization of the compound was nearly identical with gephyrin in costaining experiments ( figure  s3 g ) .
next , we asked whether gephyrin was  functionally required for the insulin increase in artemether - treated tc1 cells . using a specific small hairpin rna ( shrna ) targeting gephyrin
, we achieved a > 70% reduction of gephyrin protein in tc1 cells ( figure  s3h ) .
in contrast to control - treated   cells , cells treated with an shrna targeting gephyrin ( shgephyrin ) failed to increase intracellular insulin levels after artemether treatment ( figure  2d ) .
single - cell analysis of the imaging data revealed a high correlation between the increases of gephyrin and insulin in artemether - treated cells , which was largely abolished when gephyrin was knocked down ( figure  2e ) .
gephyrin exerts different functions , including enzymatic activity in the synthesis of the molybdenum cofactor moco , regulation of mtor signaling via direct interaction with mtor ( mechanistic target of rapamycin ) ( sabatini et  al . , 1999 ) , and structural roles in the transport of glycine and gabaa receptors to the membrane ( fritschy et  al . , 2008 ) .
we observed increased moco synthesis in artemether - treated  cell extracts , indicative of enhanced gephyrin enzymatic activity ( figure  s4a ) .
moreover , a small fraction of endogenous gephyrin interacted with mtor , and artemether inhibited this interaction  ( figure  s4b ) . finally , several subunits of the gabaa - receptor complex , which physically interacts with gephyrin , were increased on both the mrna and protein levels following artemether treatment ( figures 3a and 3b ; figure  s3f ) .
rna sequencing ( rna - seq ) experiments further underlined the effects of artemether on gabaa - receptor signaling in tc1 cells .
gene ontology ( go ) term enrichment analysis identified synaptic transmission among the pathways significantly altered by artemether ( figure  3c ) , and we observed the upregulation of the genes p2rx3 , vamp1 , and nrxn3 involved in the gaba - signaling pathway ( figure  s4c ) .
therefore , we used 3-day pre - incubation with 10  m artemether to test whether adaptive cellular responses , such as gabaa - receptor - mediated effects , could contribute to changes in tc1 cell excitability to modulate hormone release .
artemether - treated cells exhibited significantly lower basal ca levels ( figures 3d and 3e ) , including coincidently reduced nuclear ( figure  s4d ) and cytoplasmic ( figure  s4e ) ca relative to control cultures . likewise , partial substitution of extracellular na by k ( to trigger excitation ) induced differential responses in drug - exposed tc1 cells : physiologically relevant 15  mm kcl increased the fraction of cells  with ca transients , suggesting increased excitability , as compared to untreated controls ( figure  3f ) . in 50  mm kcl , which induces signal saturation due to maximal ca influx through voltage - gated ( vg ) ca channels , artemether - treated cells showed attenuated slow ca responses in the second timescale ( figure  3 g ) , which was mimicked by pre - treatment of control cells with the gabaa - receptor antagonist picrotoxin .
next , we tested whether acute application of muscimol , a gabaa - receptor agonist , could potentiate kcl ( 15  mm)-induced responsiveness and whether it exhibited an additive effect with artemether . indeed , muscimol - exposed cells showed further increases in their intracellular ca when simultaneously exposed to kcl .
pre - incubation with the gabaa - receptor antagonist picrotoxin abolished these effects ( figure  3h ) , confirming gabaa - receptor involvement . based on these results , we suggest that artemether pre - treatment resets intracellular ca through gabaa - receptor - mediated mechanisms and acutely modulates tc1 cell excitability by potentiating gabaa - receptor signaling .
moreover , the data show that gaba can act as an excitatory stimulus in tc1 cells .
since gabaa receptors gate cl ions , we sought to directly monitor intracellular cl concentrations by using mqae dye imaging .
3-day pre - treatment with artemether induced an increase in intracellular cl ( figure  3i ) , which was not further changed when treated with muscimol acutely ( figure  s4f ) .
similarly , we detected muscimol - induced transmembrane currents at the low - picoampere range , if any , in both control and in artemether - treated tc1 cells acutely using perforated patch recordings ( data not shown ) .
even though this might be seen as contradictory to a mechanism reliant on gabaa - receptor - gated currents , we argue that the high input resistance ( > 1 g ) of all recorded cells , together with the presence of vg ca currents operating at the picoampere range ( figure  s4 g , pink shading ) , and with high - amplitude delayed - rectifier k channels ( figure  s4
g ) , might be sufficient to induce membrane potential oscillations because of the initial activation of vg ca channels ( depolarization ) followed by the activation of vg k channels ( repolarization ) ( figure  s4h ) . accordingly , the injection of minute currents ( up to 5  pa ) in current - clamp mode was sufficient to induce quasi - action potentials , which we expect to be responsible for the generation of ca transients regulating exocytosis .
thus , artemether treatment can evoke global differences in tc1 cell excitability and hormone release through the potentiation of functional gabaa receptors . to prove that gabaa - receptor signaling plays a functional role in  the mechanism of action of artemisinins in tc1 cells , we combined artemether with structurally and mechanistically distinct gabaa - receptor antagonists ( figures 3j and s4i ) . while these  compounds had different effects on baseline insulin expression by themselves , all compounds partially inhibited the effects of artemether on insulin expression .
we next tested whether treatment with different gabaa - receptor agonists alone was  sufficient to induce insulin expression in tc1 cells ( figure  3k ) .
most compounds  particularly , the positive allosteric modulators etifoxine and ns11394induced insulin expression , but all gaba - receptor agonists were less potent relative to artemether .
gaba is normally co - secreted with insulin by  cells , and suppressing glucagon secretion is one of the physiological functions of gaba signaling in  cells ( li et  al . , 2013 ,
, 1989 , wendt et  al . , 2004 , xu et  al . , 2006 ) . in tc1 cells , artemether treatment inhibited glucagon secretion in low - glucose conditions ( figure  4a ) .
interestingly , supplementing recombinant glucagon abolished the effect of artemether on insulin induction ( figure  4b ) .
these data imply that maintaining the correct concentration of glucagon in close vicinity to  cells is critical for  the stability of this cell type . in order to lower the extracellular glucagon concentration , we applied decanoyl - rvkr - cmk ( pci ) , a highly potent , irreversible , cell - permeable inhibitor of proprotein convertases that inactivates prohormone convertase 2 at subnanomolar concentrations ( denault et  al . , 1995 ) .
treatment of tc1 cells with this compound resulted in a dramatic increase of intracellular glucagon staining , consistent with the accumulation of unprocessed proglucagon ( figure  4c ) . at the same time , these  cells stained positive for insulin protein ( figure  4c ) . on the transcriptional level , the decrease of glucagon secretion repressed glucagon expression and significantly upregulated the expression of  cell - specific transcription factors pax4 and mnx1 ( figure  4d ; table s2 ) .
these results suggest that artemether - mediated amplification of gaba - receptor signaling prevents glucagon secretion , thereby reducing extracellular glucagon and triggering the loss of  cell identity . to characterize the physiological relevance of our findings , we applied artemether in  vivo in three model organisms . for developmental effects of the compound
, we studied zebrafish carrying a gfp transgene under the glucagon promoter and an mcherry construct controlled by the insulin promoter .
5-day treatment of zebrafish larvae with artemether altered islet morphology and caused a dose - dependent reduction in  cell numbers , with   cell numbers affected at higher concentrations ( figures s5a and s5b ) .
next , we applied the compound in a zebrafish  cell ablation model at a later developmental stage . in animals expressing an insulin - promoter - driven caspase-8 transgene that can be activated pharmacologically
,  cells were completely ablated by the addition of a small - molecule dimerizer at 3  days post - fertilization ( dpf ) for 48  hr .
the expression of a far - red fluorophore ( e2crimson ) under the control of the insulin promoter allowed the analysis of  cell numbers at 12 dpf ( figures 5a and 5b ) .
based on the known regenerative capacity of zebrafish , control ablated animals had an average of eight reestablished  cells at this time point . in animals ablated and treated with artemether for 4  days ( 812 dpf ) , we observed a 75% increase in  cell number to 14 cells per animal on average .
next , we analyzed the effects of  cell ablation and artemether on glucose levels in zebrafish .
while  cell ablation resulted in a 34% increase in glucose levels 7  days post - ablation , artemether treatment after ablation inversed that phenotype , and animals had 42% lower glucose levels compared to unablated controls ( figure  5c ) .
in addition to these developmental models , we also sought to study artemisinin effects on mature islets in wild - type mice . in line with in  vitro data
, we observed an increase in pancreatic islet size following 3-month treatment with artesunate in drinking water ( figures 5d and 5e ) .
these changes occurred in the absence of a dramatic shift in islet subpopulations or cell proliferation ( figure  s5c ) , thereby phenocopying arx loss in  vivo ( courtney et  al . , 2013 ) .
to confirm  cells as  the source of new insulin - positive cells , we turned to a lineage - tracing model .
we isolated islets from mice harboring a construct for cre recombinase under the control of the glucagon promoter and a floxed red fluorescent protein ( rfp ) allele integrated in the rosa26 locus . thereby , all cells that have ever activated glucagon expression are permanently labeled rfp positive . in control
dmso - treated islets , we observed that only a background of approximately 1% cells were double positive for insulin and rfp .
treatment of these islets for 24  hr with artemether resulted in 4-fold upregulation of this cell population ( figures 5f and 5 g ) .
these data indicate that artemether induces a rapid induction of insulin expression in  cells within mouse islets , resulting in long - term expansion of the insulin - producing cell population and islet mass . finally , to analyze the effects of artemether treatment in a rodent diabetes model , we ablated   cells in rats with a single high - dose streptozotocin injection .
animals were randomized to treatment and control groups based on matching blood glucose measurements on day 9 post - ablation and then treated orally with artemether or control vehicle for 23  days .
after overnight fasting , baseline blood glucose levels  in artemether - treated animals were drastically reduced compared to those of control rats ( figure  5h ) .
these lower baseline glucose levels were also reflected in an oral glucose tolerance test performed in the two groups ( figures 5i and 5j ) .
we next assessed the effects of artemether on  cells in primary human pancreatic islets ( figure  6a ; table s3 ) .
the drug caused a rapid translocation of arx to the cytoplasm and a reduction in the staining intensity for this master regulatory transcription factor ( figure  6a ) .
to better visualize the artemether - induced translocation of arx to the cytoplasm , we treated intact islets and then dissociated them to single cells immediately prior to immunofluorescence staining ( figure  6b ) .
we observed that active gabaa - receptor signaling was a requirement for the translocation of arx to the cytoplasm following artemether treatment , since co - treatment with artemether and the gabaa - receptor antagonist picrotoxin maintained nuclear arx localization ( figure  6c ) .
we then studied the effect of artemether on hormone levels and gene expression in primary human islets .
compared to control dmso - treated islets , artemether increased the number of double - positive cells expressing both insulin and glucagon ( figures s6a  s6c ) .
the effects of artemether or gaba on global transcriptional profiles were tested in bulk samples containing 500 cells .
gene set enrichment analysis ( gsea ) revealed significantly decreased expression of exocrine - specific genes ( li et  al . , 2016 ) , both with artemether and gaba ( figure  s7a ; table s1 ) .
in contrast , genes upregulated by both artemether and gaba significantly overlapped with an endocrine , but not an exocrine , gene set ( figure  s7b ) . to investigate the  cell - type - specific gene expression changes with artemether treatment , we performed single - cell transcriptomics on human primary islets .
we treated intact islets with artemether or dmso , dissociated the islets immediately prior to sorting single cells to individual wells of a 96-well plate containing lysis buffer , and performed single - cell rna - seq as previously described ( li et  al . , 2016 ) .
we then assessed global transcriptomic alikeness between individual cells through multidimensional scaling ( figure  6d ) .
we observed that compound - treated cells clustered with dmso - treated controls , indicating that up to 72  hr of in  vitro treatment with artemether did not induce a whole new cell type .
looking for changes in specific genes related to the compounds mechanism , we found that gabrb3 and gabrg2 were strongly upregulated in artemether - treated  cells ( figure  6e ) , consistent with the findings in the mouse cell line .
an increase in gephyrin and gaba - receptor subunit protein levels on some islet cells was also evident by immunofluorescence staining ( figure  6f ) . to analyze transcriptome - wide changes , we used gsea to identify differentially regulated pathways in dmso- versus artemether - treated single - cell transcriptomes . in  cells ,
artemether downregulated some cell - type - specific transcripts ( figures s7c and s7d ) . in  cells
, we observed a strong and significant downregulation of  cell - specific genes , including eif4a1 , cryba2 , pdk4 , and muc13 following artemether treatment ( figures 6 g and 6h ) .
key  cell - specific genes were upregulated in  cells following artemether treatment , including gnas and abcc8 , the gene coding for the sulfonylurea receptor ( figures s7e and s7f ) .
we next assessed whether the reduction in  cell character and gain of  cell characteristics following artemether treatment had functional consequences in intact human islets .
first , we tested the expression of  cell factor arx ( figure  7a ) .
while we observed donor - to - donor variability of the effect size , arx expression was consistently reduced .
we then investigated whether the loss in  cell character corresponded to increased  cell properties .
normalized to total protein , artemether - treated islets contained more intracellular insulin compared to dmso - treated islets from the same donor ( figure  7b ) .
finally , we tested whether this insulin increase was functional and performed glucose stimulated insulin secretion assays .
while artemether treatment did not affect secretion in low - glucose conditions , artemether - treated islets from all donors tested secreted significantly more insulin compared to dmso - control - treated islets when challenged with high - glucose concentrations ( figure  7c ) .
we identified artemisinins as approved drugs that can confer  cell characteristics to  cells .
this phenotype depends on the stabilization of the protein gephyrin , which , in turn , activates gaba signaling in  cells .
active gaba signaling is required for the effects of artemisinins on the key  cell - fate determining transcription factor arx , which translocates to the cytoplasm following drug treatment ( figure  7d ) .
it is tempting to speculate that the changes in nuclear calcium ( and/or chloride ) signaling that we observed provide a direct link between extracellular glucagon , gaba - receptor signaling , and nuclear arx localization .
previous studies have already connected  cells key function of glucagon secretion to gaba - receptor signaling .
gaba is primarily produced by  cells , whereas several gabaa - receptor subunits are specifically expressed on  cells ( franklin and wollheim , 2004 , wan et  al . , 2015 ) .
the architecture of the vasculature in islets ensures that arterioles first contact insulin - expressing cells ( bonner - weir and orci , 1982 , stagner and samols , 1992 ) , resulting in the exposure of the downstream  cells to high local concentrations of factors secreted by  cells , including insulin and gaba .
insulin is known as a major repressor of glucagon secretion ( maruyama et  al . , 1984 ) , and
the action of the hormone is dependent on active akt and gaba signaling ( xu et  al . ,
our findings suggest that gaba signaling not only affects  cell function , as measured by glucagon secretion , but also feeds back to  cell identity .
thus , we propose an additional beneficial effect of activating gaba - receptor signaling in diabetes , beyond the modulation of the immune system and  cell proliferation previously suggested ( purwana et  al . , 2014 , soltani et  al . , 2011 ) .
a key remaining question concerns the lack of  to  cell transdifferentiation in diabetes .
why would evolution equip  cells with a high amount of epigenetic plasticity , so that loss of a single transcription factor is sufficient for their transdifferentiation , and why would a mechanism to exclude this very transcription factor from the nucleus be maintained ?
however , the only conditions known , when  cells convert into  cells , are following the genetic manipulation of these master regulatory transcription factors ( collombat et  al . , 2009 , courtney et  al . , 2013 ) and a toxin - induced model of near - complete  cell loss ( thorel et  al . , 2011 ) .
particularly in type-2-diabetic conditions , several of the factors observed to contribute to arx repression are altered in similar ways to the changes we observe with artemisinins .
for example , gaba receptors accumulate on the surface of  cells in high - glucose / high - insulin conditions ( xu et  al . , 2006 ) .
moreover , long - term treatment with gaba alone is sufficient to induce  cell - mediated , -like cell neogenesis in a mouse model ( ben - othman et  al . , 2016 ;
however , we still only have a limited understanding of the intra - islet changes associated with diabetic conditions , including local concentrations of  gaba , insulin , and glucagon , as well as the composition of gaba receptors and other factors affecting the autocrine and paracrine effects of these molecules .
insufficient  cell - derived gaba levels and inappropriate glucagon secretion in diabetic high - glucose conditions could , therefore , prevent endogenous  cell transdifferentiation in diabetic conditions . in our  cell - line model ,
likely , the strong transcriptional induction of several gaba - receptor subunits following drug  treatment induces a specific gaba - receptor subunit composition not normally present in  cells .
these receptors then increase gaba responsiveness and alter baseline calcium signaling of artemether - treated  cells , and the effects could potentially synergize with other aspects of gephyrin biology and pi3k signaling .
artemisinin combination therapy is the treatment of choice for malaria and more than 300 million doses are dispensed annually ( dondorp et  al . , 2010 ) . despite this large patient cohort ,
no clinical data on the effects of artemisinins on human pancreatic endocrine function are available .
first , the acute life - threatening condition of plasmodium - infected patients , together with the known propensity of plasmodium infection to cause hypoglycemia , makes blood glucose levels highly variable in the short term .
second , in healthy individuals , a small increase in  cell number is not expected to cause a phenotype , as these cells secrete insulin in a highly glucose - regulated manner .
unfortunately , currently no imaging methods are available to directly assess human  cell mass .
we observe rapid effects of these compounds on cell lines , isolated islets , and zebrafish larvae in the timescale of hours to days .
in contrast , effects in rodent models take weeks to months , potentially because of the lower bioavailability of artemisinins in  vivo .
plasma half - lives of these compounds are in the range of minutes to a few hours , and the poor solubility in aqueous solutions precludes the continuous dosing using pumps ; hence , we rely on  once - daily oral dosing in rats . in contrast
, for malaria patients , a  typical treatment cycle with artemisinins lasts only 4  days , but  dosing occurs at relatively high concentrations every 8  hr
. currently , clinical studies are ongoing for the long - term effects  of artemisinins in cancer patients ( ericsson et  al . , 2014 ) ( nct02263950 ) . therefore ,
investigation of the effects of long - term artemether treatment for the induction of endogenous insulin expression in c - peptide - negative type 1 diabetes patients appears clinically feasible .
regardless of the direct clinical use of artemether in diabetes , our findings open up completely new avenues for transdifferentiating  cells by structurally different gephyrin stabilizers , gaba - receptor transcriptional activators , and additional targets in the  gaba - receptor signaling pathway , toward a treatment for type 1 diabetes .
reagent or resourcesourceidentifierantibodiesinsulinsigmacat#18510dakocat#a056401 ; rrid : ab_2617169linkocat#4011 - 01glucagonabcamcat#ab92517 ; rrid : ab_10561971arxr&dcat#af7068 ; rrid : ab_10973178myc - tagcell signaling technologycat#2276histone h2bcell signaling technologycat#2934gephyrinabcamcat#ab25784 ; rrid : ab_1209349gephyrin ( figure  s4b)synaptic systemscat#147111 ; rrid : ab_887719gaba - receptor subunit beta 3/4milliporecat#mab341 ; rrid : ab_2109419gaba - receptor subunit alpha 2abcamcat#ab176170gaba - receptor subunit gamma 2abcamcat#ab101036 ; rrid : ab_10672116mtorcell signaling technologycat#2972p - mtorcell signaling technologycat#5536aktcell signaling technologycat#4691p - aktcell signaling technologycat#4060p ; rrid : ab_972551actinsigmacat#a2066 ; rrid : ab_476693alpha - tubulinabcamcat#ab7291 ; rrid : ab_2241126dsredclontechcat#632496 ; rrid : ab_10013483goat anti - rabbit 633thermo fischercat#a-21070 ; rrid : ab_2535731goat anti - guinea pig 546thermo fischercat#a-11074 ; rrid : ab_2534118goat anti - guinea pig 488thermo fischercat#a-11073 ; rrid : ab_2534117donkey anti - rabbit 488thermo fischercat#a-21206 ; rrid : ab_2535792donkey anti - mouse 488thermo fischercat#a-21202 ; rrid : ab_2535788donkey anti - sheep 546thermo fischercat#a-21098 ; rrid : ab_2535752donkey anti - rabbit 546thermo fischercat#a-10040/a-10036/a-10074 ; rrid : ab_2534016rabbit anti - sheep igg - hrpsanta cruzcat#sc-2770 ; rrid : ab_656968donkey anti - rabbit hrpjackson immunoresearchcat#711 - 035 - 152 ; rrid : ab_10015282donkey anti - mouse hrpjackson immunoresearchcat#715 - 035 - 151 ; rrid : ab_2340771donkey anti - sheep hrpjackson immunoresearchcat#713 - 035 - 147 ; rrid : ab_2340771donkey anti - goat hrpjackson immunoresearchcat#705 - 035 - 147 ; rrid : ab_2313587chemicals , peptides , and recombinant proteinsartemethersigmacat#a9361artemether ( figures 5h5j)cayman europecat#11815bicucullinetocriscat#0130gabazinesigmacat#s106picrotoxinsigmacat#p1675salicylidene salicylhydrazidesigmacat#s8194etifoxinesigmacat#sml0272muscimolsigmacat#m1523vgabatrinsigmacat#v8261halothanemedchemexpresscat#hy - b1010ns11394medchemexpresscat#hy-11048zolpidemsigmacat#e006thapsigarginsigmacat#t9033gaboxadolsanta cruzcat#sc204342furosemidetocriscat#3109fg-7142sigmacat#e006elesclomolselleckcat#s1052nacetylcysteinesigmacat#a9165lynxtag_artbiolynx technologiescat#bl007ruopcienzo life sciencescat#alx-260 - 022-m001artesunatesigmacat#a3731artesunate ( figures 5d and e)tcicat#a2191deoxyarteethersanta cruzcat#sc-211235arteethersanta cruzcat#sc-217662n - hydroxy-11-azaartemisinintrccat#h804500artemisitenetrccat#a777550artemisonealsachimcat#c1740artemisitenalsachimcat#s1873dihydro artemisinintrccat#a637875recombinant glucagonsigmacat#g2044dimerizer ap20187clontechcat#635059pe2-crimson - n1 vectorclontechcat#632554methylcellulosesigmacat#m6385hoechst 33342thermo fischercat#h1399critical commercial assaysxanthine oxidase assay kitabcamcat#ab102522pi(3)p mass elisa kitecheloncat#k-3300glucagon elisaalpcocat#48-gluhu - e01-alinsulin elisaalpcocat#80-inshu - e01.1glucose assay kitbiovisioncat#k606 - 100deposited datasingle cell rna - seq of the control cellsncbi gene expression omnibusgeo : gse73727single cell rna - seq of the artemether treated cellsncbi gene expression omnibusgeo : gse84714rna - seq of bulk samplesncbi gene expression omnibusgeo : gse84592experimental models : cell linestc1novo nordiskatcc cat#crl-2934tc1-ltthis papern / amin6novo nordiskmiyazaki et  al . , 1990min6-arxthis papern / aexperimental models : organisms / strains(gcga : gfp)- ( ins : ntr - mcherry ) zebrafishthis papern / ains : caspase8;ins : e2crimson zebrafishthis papern.s . and d.m . , unpublished dataglucre::rfp mousethis papern / arecombinant dnatetr - cmv - arx - myc - flagthis papern / asv40 large t - antigen lentivirusgentargetcat#lvp016-neosequence - based reagentsprimers for ins2 , actb , pax4 , actbfomina - yadlin et  al . , 2010n / aprimers for mnx1 , gcg , arxthis papertable s2shgephyrinsigmacat#trcn0000098585shcontrolsigmacat#shc001software and algorithmstophat v2.0.4kim et  al . , 2013https://ccb.jhu.edu / software / tophat / index.shtmlrseqc packagewang et  al . , 2012http://rseqc.sourceforge.net / gene set enrichment analysissubramanian et  al . , 2005http://software.broadinstitute.org / gsea / downloads.jspmootha et  al .
2012https://fiji.sc / cell counter pluginkurt de vos , university of sheffield , academic neurologyhttps://imagej.nih.gov / ij / plugins / cell - counter.html further information and requests for reagents may be directed to , and will be fulfilled by the lead contact stefan kubicek ( skubicek@cemm.oeaw.ac.at ) .
tc1 cells were purchased from atcc ( crl-2934 ) and grown in low - glucose dmem supplemented with 10% fbs , 50  u / ml penicillin and 50  g / ml streptomycin .
min6 cells with doxycycline - inducible constructs were grown in high - glucose dmem supplemented with 15% tet system approved fbs ( clonetech 631106 ) , 71  m 2-mercaptoethanol , 50  u / ml penicillin and 50  g / ml streptomycin .
the mouse islets from the  cell lineage - tracing animals were kept in rpmi medium supplemented with 10% fbs , 50  u / ml penicillin and 50  g / ml streptomycin .
the cell culture for human islets followed established protocols ( walpita et  al . , 2012 ) .
all mouse work was conducted in patrick collombat s lab as previously described ( courtney et  al . ,
2013 ) according to french ethical regulations under the animal license nce/2012 - 85 and nce/2014 - 190 .
rat experiments were conducted at physiogenex according to the standard operating procedure in place at the test facility and an agreement from the french veterinary services .
all procedures were performed in accordance with the guide for the care and use of laboratory animals ( revised 1996 and 2011 , 2010/63/eu ) and french laws .
transgenic ( gcga : gfp)-(ins : ntr - mcherry ) zebrafish were maintained according to the guidelines of the local authorities under licenses gz:565304/2014/6 and gz:534619/2014/4 .
transgenic ins : caspase8;ins : e2crimson ( abbreviated ins : casp8 ) zebrafish embryos were generated under license bmwf-66.008/0017-ii/3b/2013 .
human islets were obtained from the european consortium for islet transplantation ( geneva ) and the integrated islet distribution program iidp .
this study is approved by the ethics committee of the medical university of vienna ( ek - nr .
detailed donor information is provided in table s3 . to generate inducible min6 cells , we first transduced cells with a lentivirus carrying tetr dna and a neomycin resistance cassette ( gentarget , cat .
# lvp017-neo ) and then isolated a single clone expressing high amounts of tetr protein , validated with the tet01  antibody ( mobitec ) .
this tetr expressing cell line was then transduced with virus particles coding for flag - myc tagged ( c - terminal ) human arx cdna also expressing the puromycin resistance gene .
the vector used for this work was pstk007 , a modified version of the plenti4.2 ( life technologies ) .
then , a second round of clonal selection was performed where numerous clones were tested for maximum repression in the uninduced state and a high degree of induction upon treatment with doxycyline ( 1g / ml ) .
all lentiviruses were produced in hek293 t cells using the virapower plasmid mix ( life technologies ) , according to the manufacturer s protocol .
all virus work was done in a class ii laboratory , and before transferring the cells to a regular lab the cells were proven to be virus free using a p24 elisa kit ( cell biolabs , cat.#vpk-108-h ) . to establish the tc1 subclone used for all experiments described in this study ,
tc1 cells were infected with a lentiviral vector causing the expression of large t antigen under the cmv promoter to make them independent of the glucagon - promoter driven large t antigen expression .
50 nl compound per well was transferred to black optical suitable 384-well plate ( corning 3712 ) from dmso stock plates using acoustic transfer ( labcyte echo 520 ) .
3000 min6 cells per well were then plated in 50  l media containing 1  g / ml doxycycline on top of the compounds .
three days after seeding , cells were fixed in 3.7% formaldehyde for ten minutes at room temperature . following washing with pbs
, cells were fixed with cold pure methanol at 20c for 10  min , permeabilized with 1% triton x-100 in pbs for 30  min and blocked with 3% bsa in pbs for 30  min .
20  l primary anti - insulin antibody ( 1:2000 ) in 1.5% bsa / pbs , was added per well and incubated at 4c overnight . after washing with pbs twice ,
20  l cy-3labeled donkey--guinea pig antibody ( 1:1000 ) and 10  g / ml hoechst 3342 in pbs was added per well and incubated for 1  hr .
after two washes with pbs , plates were stored in 4c in dark until analysis .
images were taken by an automated microscope ( perkin elmer operetta ) using a 20x objective .
images were exposed for 10  ms in  the hoechst channel and for 500  ms in the alexa fluor 546 channel .
nuclei were identified ( harmony method c ) and cytoplasm was defined based on the nuclei ( harmony method c ) . in total 1152 wells
were screened containing 280 compounds from the cemm library of unique drugs ( cloud ) ( m. licciardello , p. markt , f. klepsch , and s.k . , unpublished data ) in triplicates with control wells .
hits were selected based on the intensity of insulin in the  alexa fluor 546 channel and cell numbers in the hoechst channel .
immunofluorescence staining for all other antibodies was performed as described above , images were acquired on an automated microscope ( perkin elmer operetta ) or a leica dmi6000 b inverted confocal system with a 63x 1.30 acs apo lens .
image analysis and quantitation was performed as described above . for quantification , at least 1,000 cells were analyzed for each condition .
cells were lysed and rna isolated using the rneasy mini kit ( qiagen ) according to the manufacturer s protocol .
the amount of total rna was quantified using a qubit 2.0 fluorometric quantitation system ( life technologies ) and the rna integrity number ( rin ) was determined using an experion automated electrophoresis system ( bio - rad ) .
the libraries for rna - seq after 24h induction in min6 cells were prepared with ribo - zero and scriptseq v2 kits ( epicenter ) following the provider s manual .
other rna - seq libraries were prepared with truseq stranded mrna lt sample preparation kit ( illumina ) using sciclone and zephyr liquid handling robotics ( perkinelmer ) .
library amount was quantified using the qubit 2.0 fluorometric quantitation system ( life technologies ) and the size distribution was assessed using experion automated electrophoresis system ( bio - rad ) .
libraries were pooled and sequenced on the illumina hiseq platform using the 50  bp single - read configuration .
reads were aligned with tophat ( v2.0.4 ) with the  no - novel - juncs  no - novel - indels options ( kim et  al . , 2013 ) .
gene expression was calculated as reads per kb per millions of reads ( rpkms ) using rpkm_count.py from the rseqc package ( wang et  al . , 2012 ) and the ncbi rna reference sequences collection ( refseq ) downloaded from ucsc .
sample preparation and data analysis for single cell rna - seq was conducted as previously described ( li et  al . , 2016 ) .
after the rna was isolated with the rneasy mini kit ( qiagen ) , it was reverse transcribed with random primers using the high capacity cdna reverse transcription kit ( applied biosystems ) .
quantitative pcr was performed with power sybr green pcr master mix ( applied biosystems ) on a lightcycler 480 ( roche ) .
ratiometric imaging was performed using a visichrome monochromator ( visitron systems ) on a coolsnap hq2 camera ( photometrics ) and recorded in visiview software ( visitron systems ) .
krebs - ringer solution was used as basic external solution and contained : 119  mm nacl , 2.5  mm kcl , 1  mm nah2po4 , 1.5  mm cacl2 , 1.5  mm mgcl2 , 20  mm hepes , 11  mm glucose ( ph  7.4 ) .
a p value of  < 0.05 was considered statistically significant , and calculated by the mann - whitney rank sum test ( unpaired test ) or wilcoxon s signed rank test ( paired test ) , as appropriate .
tc1 cells were assayed by patch - clamp electrophysiology using the gramicidin or amphotericin perforated - patch configuration .
ionic currents and membrane potential were recorded , filtered , and analyzed using an axopatch 200 b amplifier , digidata1322 interface , and the pclamp8 software ( molecular devices ) .
the external solution contained : 119  mm nacl , 2.5  mm kcl , 1  mm nah2po4 , 1.5  mm cacl2 , 1.5  mm mgso4 , 11  mm glucose and 20  mm hepes - naoh ( ph 7.4 ) .
for the measurement of muscimol - induced currents the following intracellular solution was used : 140  mm cscl , 4  mm nacl , 2  mm mgcl2 , 10  mm hepes , 1  mm egta , 4  mm atp - na2 , 400  mg / l amphotericin , ( ph adjusted to 7.3 with naoh ) . for studying excitable properties of tc1 cells
the intracellular solution contained : 114  mm k - gluconate , 6  mm kcl , 10  mm hepes , 5  mm egta , 4  mm atp - mg , 30 - 50  mg / l gramicidin ( ph  adjusted to 7.3 with koh ) .
muscimol induced cl changes , tc1 cells were preincubated with mqae for 0.5  hr ( thermo fisher scientific ) . the fluorescence was recorded with coolsnap hq2 camera ( photometrics ) and visiview software ( visitron systems ) using 350  nm excitation with visichrome monochromator ( visitron systems ) and the emission filter ( 445  50  nm ) .
basal levels were measured after 1  hr incubation with mqae and washing with pbs by live cell imaging on an automated microscope ( perkin elmer operetta ) using a 20x objective and analyzed using the harmony software ( perkin elmer ) .
the measurement of moco synthase was performed with xanthine oxidase assay kit ( abcam ab102522 ) .
whole cell extracts were generated by lysing cells in np-40 buffer containing 150  mm sodium chloride , 1.0% np-40 and 50  mm tris , ph 8.0 supplemented by protease inhibitor cocktail ( roche ) .
30  g whole cell lysates were loaded onto a sds  polyacrylamide gel for electrophoresis at 30 ma per gel , and then transferred by electrophoresis to a nitrocellulose membrane ( ge healthcare life science ) .
all the blots were incubated with corresponding primary antibodies diluted 1:1000 in 5% milk at 4c overnight followed by hrp - labeled secondary antibodies ( 1:20000 ) for 1  hr at room temperature .
primary antibody was incubated with dynabeads protein g ( life technologies 10765583 ) for 15  min at room temperature and washed with pbs  containing 0.02% tween-20 ( pbst ) .
antibody - coupled beads were then incubated with tc1-lt cell lysates overnight at 4c , washed with pbst for three times and eluted with sds - loading buffer .
pi3p measurements were performed using the pi(3)p mass elisa kit ( k-3300 , echelon ) . in brief
, tc1-lt cells were pretreated with artemether for three days , followed by lipid extraction and an elisa assay according to the manufacturer s guideline .
cells were first incubated in high glucose medium ( 5 g / l glucose in  krbs medium ) for one hour followed by incubation in low glucose medium ( 0.5 g / l glucose in krbs medium ) for another hour .
supernatant after low glucose challenge was collected to measure glucagon content using a glucagon elisa ( alpco 48-gluhu - e01-al ) .
transgenic zebrafish strains tg(gcga : gfp ) and tg(ins : ntr - mcherry ) were used to demarcate  cells by gfp expression and   cells by mcherry expression .
double transgenic zebrafish were treated with indicated concentrations of artemether added to e3 medium from 26 hpf until 100 hpf .
zebrafish were embedded for confocal microscopy as described previously ( distel and koster , 2007 ) . z stacks of the pancreatic islets
were taken on a confocal microscope ( leica tcs sp8 x wll ) using a 25x water objective . using the measurement counting tool of the leica software on 3d models of the islets gfp - positive cells and mcherry - positive cells
transgenic lines were generated using tol2 mediated transgenesis ( kawakami et  al . , 2004 ) .
1 kb of the insulin promoter ( huang et  al . , 2001 ) was cloned into the p5e - mcs plasmid of the tol2 gateway system ( kwan et  al . , 2007 ) using the bamhi and hindiii restriction sites .
2005 ) ( a kind gift of philipp scherer ) was cloned downstream of the ins promoter in the p5e - ins plasmid .
e2crimson ( pe2-crimson - n1 vector , clontech ) was cloned into the pme - mcs plasmid of the tol2 system and expressed via a separate insulin promoter inserted via the bamhi and hindiii sites .
final constructs were generated by an lr recombination reaction as described ( kwan et  al . , 2007 ) in pdesttol2pa , and injected together with transposase mrna into fertilized eggs to generate tg(ins : caspase8;ins : e2crimson ) , abbreviated ins : casp8 , fish . zebrafish ( danio rerio ) were maintained according to standard protocols .
transgenic lines were generated and maintained in the mitfab692/b692/ednrb1b140/b140 background ( a kind gift of wolfgang driever at the university of freiburg ) .
3  days post fertilization ( dpf ) old embryos were incubated in 2  m dimerizer ap20187 ( dim ) for 48  hr at 28c in the dark .
dim was added to eggwater ( 0.3 g / l coral pro salt ( red sea ) to reach the final concentration .
control embryos were exposed to corresponding amounts of ethanol . at 5 dpf ( 0  days post ablation , dpa ) , larvae were rinsed in egg water and transferred to fresh egg water at a density of 20 fish/200  ml .
starting at 3dpa , ablated larvae were treated with either 5  m artemether or an equivalent volume of dmso for 4 d. compound was renewed every second day .
larvae were harvested at 7 dpa and fixed for 1 to 2  hr at room temperature in 4% pfa in pbs , then washed 3x 5  min with 1 x pbs/0.2% triton x-100 . to improve access of antibodies to internal structures , head and tail were cut off and the gut was cut open .
larvae were incubated in blocking buffer containing 1% dmso , 1% sheep serum , 1% bsa and 1% triton x-100 in 1 x pbs for at least 60  min at room temperature .
the embryos were then incubated overnight at 4c with primary antibody recognizing e2crimson ( rabbit anti - dsred , 1:100 dilution ) , washed and then reblocked and incubated in secondary antibody ( anti - rabbit alexa fluor 633 conjugated , 1:1000 dilution ) overnight at 4c .
confocal images were acquired using a zeiss lsm exciter5 microscope with a 40x water objective . for glucose assays ,
after removal of all liquid , larvae were resuspended in 200 l ice - cold , autoclaved 1x pbs , then sonicated on ice with 6 pulses of 0.5 s , and centrifuged at 13,000 rpm for 15  min at 4c .
the glucose assay was performed as described ( kimmel et  al . , 2015 ) on biological replicates , using 15 l larvae
sample buffer was added up to 25  l , and combined with 25 l reaction mix .
after incubation at 37c for 30  min , ex / em 535/590 was read on a microplate reader .
protein concentration ( a280 ) was measured on a nanodrop and used to normalize glucose concentration to protein content .
insulin - expressing ( e2crimson+/alexa 633 + ) cells were counted in z stacks using the cell counter plugin of imagej .
confocal images were processed by a median filter in imagej to remove speckle noise and assembled using adobe illustrator .
/ l glucose in krbs medium ) for one hour and in high glucose medium ( 5 g / l glucose in krbs medium ) for another hour .
supernatant was collected after low and high glucose incubations to measure insulin content using an elisa kit ( alpco 80-inshu - e01.1 ) .
the islet pellets after high glucose incubation were lysed in np-40 buffer and intra - islet insulin content was measured using the same kit .
two months old 129/sv mice were treated with artesunate ( 1mg / ml ) in drinking water provided ad libitum for 3  months .
animals were sacrificed by cervical dislocation and the tissues were fixed and sectioned as previously described ( collombat et  al . , 2003 ) . for the ex  vivo lineage tracing experiments , islets from 2  months old glucre::rfp animals ( containing the following transgenes : the rat glucagon promoter upstream of the phage p1 cre recombinase ( herrera , 2000 ) and the rosa26 promoter upstream of a loxp - stop - loxp - tdrfp cassette ( luche et  al . , 2007 ) ) were isolated by collagenase type xi ( 1  mg / ml ) digestion followed by a density gradient to purify the islets from the acinar tissue . approximately 250 islets per group were handpicked under the microscope and treated with either 10  m artemether or dmso as control . following 24 h of treatment ,
the islets were dissociated , fixed , and plated into a 384-well  plate . following washing with pbst and blocking with bsa
, the cells were incubated with guinea pig polyclonal anti - insulin ( 1/500-linco ) for 2 h at room temperature .
the cells were then washed and incubated with 488-alexa anti - guinea pig ( 1/1000-molecular probes ) together with the nuclear counter - stain dapi for 1 h. the cells were then imaged on an automated microscope ( perkin elmer operetta ) using a 20x objective and analyzed using harmony software ( perkin elmer ) .
rat experiments were conducted on 2  months old male sd rats which were fasted overnight and treated with 60  mg / kg streptozotocin i.p . on day 9
post ablation animals were randomized to treatment and control groups based on matching fasting glucose levels , and treated with 20  mg / kg artemether p.o . for 7 
16 d. 1% methylcellulose was used as vehicle , prepared once per week by dissolving in distilled water , heating to 40c , then stored at 4c .
blood glucose were measured via tail nick using a handheld glucometer ( accu - check performa roche diagnostics ) .
nmr spectra were recorded on a bruker avance iii 400 ( bruker , billerica , ma , u.s ) .
mass spectra were recorded using a xevo - uplctq - ms system ( waters , milford , ma , u.s . ) .
purification by flash column chromatography ( fcc ) was performed using silica gel 60 ( merck , darmstadt , germany ) , mplc was performed on a biotage isolera system ( biotage , uppsala , sweden ) .
drug - affinity matrices were prepared essentially as described previously ( fernbach et  al . , 2009 ,
2.7  l , 40  mol ) , ethanolamine ( 9.7  l , 160  mol ) , and trimethylamine ( 15  l , 108  mol ) were added to 500  l nhs - activated sepharose 4 fast flow beads ( ge healthcare bio - sciences ab , uppsala , sweden ) and the reaction was incubated on a rotary shaker for 24  hr . beads were washed and resuspended in dmso and nhs - activated artesunate ( 100  l , 1.00  mol ) was added to the suspension , and the mixture was incubated on a rotary shaker for 24  hr .
unreacted beads were blocked by addition of  nhs - acetate ( 10  mol ) and triethylamine ( 25  l , 180  mol ) , followed by agitation on a rotary shaker for 24  hr . after washing with dmso and lysis buffer , beads were incubated with cell lysates .
affinity chromatography and elution were performed in duplicate as reported previously ( fernbach et  al . , 2009 ,
after elution , enriched proteins were reduced with dithiothreitol , cysteine residues alkylated by incubation with iodoacetamide and the samples digested with modified porcine trypsin ( promega , madison , wi ) .
three percent ( and multiples thereof ) of the digested eluates were purified and concentrated by c18 reversed - phase material .
samples were analyzed at ph 2.4 on an agilent 1200 nano - hplc system ( agilent biotechnologies , palo alto , ca ) coupled to a hybrid linear trap quadrupole ( ltq ) orbitrap velos mass spectrometer ( thermofisher scientific , waltham , ma ) utilizing the xcalibur software version 2.1 .
the 15 most intense ions were isolated and fragmented by collision - induced dissociation ( cid ) .
dynamic exclusion for selected ions was 60  s and a single lock mass at m / z 445.120024 ( si(ch3)2o)6 ) was used for internal mass calibration ( olsen et  al . , 2005 ) with the target loss mass abundance of 0% .
maximal ion accumulation time allowed was 500  ms and overfilling of the c - trap was prevented by automatic gain control set to 10 ions for a full ftms scan and 5   10 ions for msn .
intact peptides were detected in the orbitrap mass analyzer at a resolution of 60,000 with a signal threshold of 2,000 counts for triggering an ms event .
further details of the lcms methodology are as previously described ( maurer et  al . , 2013 ) .
peak extraction and conversion of raw ms files into the mgf format was performed with msconvert ( proteowizard library v2.1.2708 ) and the peak lists searched against the mouse swissprot database v2013.01_20130110 ( 24,615 sequences including isoforms obtained from varsplic.pl ( kersey et  al . , 2000 ) and appended known contaminants ) with the search engines mascot ( v2.2.03 , matrixscience , london , uk ) and phenyx ( v2.5.14 , genebio , geneva , switzerland ) ( colinge et  al . , 2003 ) .
submission to the search engines was via a perl script that performs an initial search with relatively broad mass tolerances ( mascot only ) on both the precursor and fragment ions ( 10 ppm and  0.6 da , respectively ) .
high - confidence peptide identifications are used to recalibrate all precursors and fragment ion masses prior to a second search with narrower mass tolerances ( 4 ppm and  0.3  da for cid ) .
carbamidomethyl cysteine and oxidised methionine were set as fixed and variable modifications , respectively . to validate the proteins , mascot and phenyx output files
proteins with  2 unique peptides above a score t1 , or with a single peptide above a score t2 were selected as unambiguous identifications .
the thresholds t1 , t2 , and t3 for each search engine were set by the target - decoy method to control the false discovery rate ( fdr ) of  < 1% for protein identifications and 
for mascot , the following thresholds were used : t1  = 14 , t2  = 40 and t3  = 10 ; phenyx thresholds were set to 4.2 , 4.75 and 3.5 , respectively ( p value  < 10 ) .
the validated proteins retrieved by the two algorithms were merged , any spectral conflicts discarded and grouped according to shared peptides .
non - specific binders were filtered from the drug pull - downs using the saint software ( version 2.3.4 ) ( choi et  al . , 2011 ) . using protein spectral counts as a measure of protein abundance and comparing the data of from the drug pull - downs with the negative control drug competition experiments ,
we also compared saint probabilities with the fold - reduction of spectral count upon free compound competition representing a magnitude of effect .
fold - reduction was computed as the ratio of median spectral counts observed in pull - downs with / without competition . in each condition , 4 spectral counts were available for the median ( 2 biological replicates and 2 technical for each ) . quantitative proteomic analysis was performed on tc1 cells harvested after three day compound treatment and lysed with 2% sodium dodecyl sulfate ( sds ) .
samples were centrifuged at 5,000 r.c.f . for 2  min and the supernatant removed .
lysis buffer ( 1ml ) was added and the samples incubated for 20  min at rt .
the samples were then heated for 5  min at 99c and sonicated with a covaris sonicator to reduce viscosity .
the supernatant was collected . the protein amount in each sample was determined by using the bicinchoninic protein assay ( bca , pierce biotechnology , thermo scientific , il ) followed by an adaptation of the filter - aided sample preparation method ( fasp ) ( manza et  al . , 2005 , winiewski et  al . , 2009 ) .
samples were reduced with 100  mm dithiothreitol ( dtt ) at 99c for 5  min and transferred into vivacon 500 filter units ( vivaproducts ) .
sds containing buffer was removed from the sample by centrifugation and exchanged with 8  m urea in 100  mm tris - hcl buffer .
proteins were alkylated with 50  mm iodoacetamide and washed with 50  mm triethyl ammonium bicarbonate ( teab ) .
porcine trypsin ( promega , madison , wi ) was used to digest the proteins in an enzyme to protein ratio of 1:100 ( w / w ) . for relative protein quantitation , the set of six samples ( three untreated and three drug - treated ) were separately derivatised with tmt 6-plex reagents ( thermofisher scientific , waltham , ma ) according to the instructions provided by the manufacturer .
the samples were labeled with tmt 126 , 127 , 128 , 129 , 130 , 131 , respectively .
the labeled tryptic digests were pooled and concentrated by solid phase extraction ( spe ) ( macrospin columns 30 - 300  g capacity , the nest group ) .
samples were basified with 20  mm ammonium formate prior to injection onto a phenomenex column ( 150   2.0  mm gemininx - c18 3  m 110 ; phenomenex , torrance , ca , usa ) on an agilent 1200 series hplc ( agilent biotechnologies , palo alto , ca ) with uv detection at 214  nm .
hplc solvent a consisted of 20  mm ammonium formate , ph 10 in 5% acetonitrile and solvent b consisted of 20  mm ammonium formate , ph 10 in 90% acetonitrile .
peptides were separated at a flow rate of 100  l / min and eluted from the column with a two - step linear gradient from 0 to 70% solvent b. seventy two time - based fractions were collected , acidified , and pooled into 50 hplc vials based on the uv trace .
after removal of organic solvent in a vacuum centrifuge , samples were reconstituted to 10  l with 5% formic acid ( bennett et  al . , 2011 ) .
individual fractions were analyzed at ph 2.4 on an agilent 1200 nano - hplc system ( agilent biotechnologies , palo alto , ca ) coupled to a hybrid linear trap quadrupole ( ltq ) orbitrap velos mass spectrometer ( thermofisher scientific , waltham , ma ) utilizing the xcalibur software version 2.1 . for data acquisition .
single fractions were loaded onto a trap column ( zorbax 300sb - c18 5  m , 5   0.3  mm , agilent biotechnologies , palo alto , ca ) with a binary pump at a flow rate of 45  l / min .
loading and washing solvents were composed of 0.1% trifluoracetic acid ( tfa ) in water ( solvent a ) and 0.1% tfa in 70% methanol and 20% isopropanol ( solvent b ) .
the peptides were eluted by back - flushing from the trap column onto a 16  cm fused silica analytical column with an inner diameter of 50  m packed with c18 reversed - phase material ( reprosil - pur 120 c18-aq , 3  m , dr .
lcms solvents were composed of 0.4% formic acid ( fa ) in water ( solvent a ) and 0.4% fa in 70% methanol and 20% isopropanol ( solvent b ) .
elution was achieved with a 27  min gradient ranging from 3 to 30% solvent b , followed by a 25  min gradient from 30 to 70% solvent b and , finally , a 7  min gradient from 70 to 100% solvent b at a constant flow rate of 100 nl / min .
the 10 most intense ions were isolated and fragmented by higher - energy collision - induced dissociation ( hcd ) for peptide identification and relative quantitation of tmt reporter ions .
dynamic exclusion for selected ions was 60  s and a single lock mass at m / z 445.120024 ( si(ch3)2o)6 ) was used for internal mass calibration ( olsen et  al . , 2005 ) with the target loss mass abundance of 0% .
maximal ion accumulation time allowed was 500  ms and overfilling of the c - trap was prevented by automatic gain control set to  10 ions for a full ftms scan and 5   10 ions for msn hcd .
intact peptides were detected in the orbitrap mass analyzer at a resolution of 30,000 with a signal threshold of 2,000 counts for triggering an msms event .
the maximum ion scan time was set to 200  ms for acquiring 1 microscan at a resolution of 7,500 .
peak extraction and conversion of .raw ms files into the .mgf format was performed with msconvert ( proteowizard library v2.1.2708 ) and the peak lists searched against the mouse swissprot database v2014.03_201403131 ( 24,830 sequences including isoforms obtained from varsplic.pl ( kersey et  al . , 2000 ) and appended known contaminants ) with the search engines mascot ( v2.2.03 , matrixscience , london , uk ) and phenyx ( v2.5.14 , genebio , geneva , switzerland ) ( colinge et  al . , 2003 ) .
submission to the search engines was via a perl script that performs an initial search with relatively broad mass tolerances ( mascot only ) on both the precursor and fragment ions ( 10 ppm and  0.6 da , respectively ) .
high - confidence peptide identifications are used to recalibrate all precursor and fragment ion masses prior to a second search with narrower mass tolerances ( 4 ppm and  0.025  da for hcd ) .
carbamidomethyl cysteine and the tmt 6-plex tag on peptide n - termini and lysine residues were set as fixed modifications .
oxidised methionine was set as a variable modification . to validate the proteins , mascot and phenyx output files
proteins with  2 unique peptides above a score t1 , or with a single peptide above a score t2 were selected as unambiguous identifications .
the thresholds t1 , t2 , and t3 for each search engine were set by the target - decoy method to control the false discovery rate ( fdr ) of  < 1% for protein identifications and 
for mascot , the following thresholds were used : t1  = 12 , t2  = 45 and t3  = 10 ; phenyx thresholds were set to 5.5 , 9.5 and 3.5 , respectively ( p value  < 10 ) .
the validated proteins retrieved by the two search engines were merged , any spectral conflicts discarded and grouped according to shared peptides . using the isobar software ( breitwieser et  al . , 2011 ) ,
quantitation of the relative fold change of each protein after drug treatment ( figure  1f ) was calculated by determining the ratios of tmt reporter ion intensities .
the tmt reporter ion channels were normalized based on the protein median intensities and only peptides unique to each protein group were used for the quantitation .
the isobar software employs statistical models to calculate the fold change of each protein and the associated p value based on the ratios and statistical confidence of each individual reporter channel .
unless otherwise indicated , p values were calculated by student s t test ( graphpad prism ) and the exact values were indicated in figure legends .
gene set enrichment analysis was conducted as previously described ( mootha et  al . , 2003 , subramanian et  al . , 2005 ) .
raw data files for the rna sequencing analysis have been deposited in the ncbi gene expression omnibus under accession numbers geo : gse84592 , geo : gse73727 , and geo : gse84714 .
p.c . conceived the project and designed the study ; t.f . and j.h .- s . developed cell lines ; j.l . ,
conducted and analyzed cell biological experiments in rodent cell lines ; j.l . and c .- h.l .
, k.l.b . , and g.s .- f . performed and analyzed chemical proteomics experiments and global proteome studies ; r.a.r . , i.b .
, j.k . , m.f . , f.m.p . , t.p . , and c. bock conducted and analyzed rna - seq experiments ; a. spittler performed fluorescence - activated cell sorting ; n.s . , r.a.k .
m.c . , a.v . , f.a . , and p.c . conducted and analyzed mouse experiments ; f.b . and t.s .
 summarytype 1 diabetes is characterized by the destruction of pancreatic  cells , and generating new insulin - producing  cells from other cell types is a major aim of regenerative medicine . 
 one promising approach is transdifferentiation of developmentally related pancreatic cell types , including glucagon - producing  cells . in a genetic model , loss of the master regulatory transcription factor 
 arx is sufficient to induce the conversion of  cells to functional -like cells . here 
 , we identify artemisinins as small molecules that functionally repress arx by causing its translocation to the cytoplasm . 
 we show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of gabaa receptor signaling . 
 our results in zebrafish , rodents , and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic  cell mass from  cells . 
Introduction
Results
Discussion
STARMethods
Author Contributions