ionic calcium ( ca ) controls multiple cellular signaling processes in all eukaryotic cells , including proliferation , gene expression , and neurotransmitter release .
ca - binding proteins such as calmodulin play a pivotal role in ca signal transmission and amplification .
increases in the concentration of intracellular ca activate specific protein targets , among which the ca / calmodulin - dependent protein kinase ii ( camkii ) is a critical signal mediator . in response to an increase in intracellular ca , thr-286 of camkii
the coupling of ca - calmodulin to one of the camkii subunits allows for the phosphorylation of an adjacent subunit at thr .
calmodulin trapping  , confers ca - calmodulin - independent kinase activity to the complex and thus prolongs the ca signal .
thus , calmodulin trapping represents a molecular mechanism of memory , which is defined as the capacity to acquire , store ( consolidate ) , and retrieve ( evocate ) information .
camkii is a major synaptic protein that is activated during the induction of long - term potentiation ( ltp ) by ca influx through n - methyl - d - aspartate ( nmda ) receptors .
calmodulin trapping allows camkii to remain activated long after the initial ca signal has dissipated , suggesting that camkii is a memory molecule crucial for ltp .
consistent with this notion , camkii - null mice present with impaired memory formation , and camkii is essential for genesis and maintenance of ltp in postsynaptic neurons .
following presynaptic stimulation , camkii is activated in postsynaptic neurons , which creates a physiological imprint of the initial ca signal , and increases translocation of nmda receptors to the plasma membrane .
because of its capacity to remain activated long after the initial pulse of ca signaling , camkii perpetuates ca effects and modulates gene expression and the epigenetic profile of postsynaptic neurons .
camkii also participates in glucose - stimulated insulin secretion ( gsis ) , as multiple insulin secretagogues increase camkii activity . in the perfused rat pancreas ,
the dynamics of camkii activation correlate with the amplitude of gsis , and camkii activation is temporally associated with insulin secretion .
camkii is essential for appropriate gsis and is involved in several steps of this process , including the synthesis of insulin granules , the modulation of cytoplasmic content of atp , and the activation of synapsin .
importantly , camkii also regulates transcription factors central for  - cell function such as creb   and mafa . here
, we investigated whether pancreatic  - cells , like neurons , acquire and store the information contained in calcium pulses as a form of  metabolic memory  .
indeed , we find that  - cells retain memory of prior activation , and describe the molecular mechanism that contributes to this memory .
min6 insulinoma cells or mouse or human pancreatic islets were distributed into three groups : control , pulse , and pulse  +  kn93 ( 10  m ) .
initially , all groups were maintained in the same medium with 5.6  mm glucose for 24  h. after this acclimation period , the control group was exposed to 3-mm glucose for 24  h , while the pulse and pulse  +  kn93 ( 10  m ) groups were exposed to 30  mm of glucose for four 1-h  periods intercalated with 7-hour periods of 3  mm glucose .
kn93 ( a camkii inhibitor ) at 10  m was only present during the 30  mm glucose pulses .
all groups were then maintained at 3  mm glucose for a 24-h consolidation period .
insulin secretion of min6 cells   and mouse pancreatic islets   was conducted following standard procedures , and insulin was measured by ria .
one hundred and fifty islets were handpicked under a light microscope and pre - incubated for 1  h  in krbb ( krebs bicarbonate buffer ) containing 0.3 
the medium was discarded and the islets incubated for an additional hour in 500-l krbb containing 2.8 or 16.8  mm glucose .
subsequently , the supernatant was collected to evaluate insulin secretion by ria . for perifusion assays ,
150 treated human islets were placed into a perifusion chamber ( millipore , billerica , ma , usa ) . a computer - controlled fast - performance hplc system allowed programmable rates of flow and concentrations of the appropriate solutions held in a 37  c water bath .
islets were perfused for 80  min  with krbb in the absence of glucose , followed by krbb with increasing concentrations of glucose ( glucose was ramped from 0 to 30  mm at 0.75  mm / min ) . at the end of each experiment , islets were tested for maximal insulin secretion by adding 30  mm kcl to the perifusate .
samples were collected at 2  ml / min and insulin content was determined by using ria .
statistical analyses were performed using student 's t - test or a two - way anova with a bonferroni 's posttest , as required . statistical significance was set at p  <  0.05 .
in order to investigate metabolic memory of insulin - producing  - cells , we first had to develop a paradigm in which such memory was evident .
as detailed below , we established that exposure of cultured min6  - cells to four 1-h  pulses of high glucose , interspersed with 7-h  intervals of low glucose and thus mimicking postprandial glucose spikes in  vivo , were able to elicit robust metabolic memory . the detailed experimental paradigm is outlined in figure  1a .
as shown in figure  1b , min6  - cells exposed to the glucose pulse regimen showed higher insulin secretion both when exposed to low ( 2.8  mm ) or high ( 16.8  mm ) glucose than  - cells , which had been cultured continuously at low glucose .
this increase in insulin secretion was accompanied by a significant rise in the levels of phosphorylated camkii , consistent with the notion that camkii might be a molecular mediator of this metabolic memory ( figure  1c and d ) .
importantly , both the increases in basal and stimulated insulin secretion in the glucose pulse group were abolished by kn93 ( figure  1b ) , a specific camkii inhibitor .
next , we evaluated the kinetics of the  - cell response to acute glucose exposure , and observed that the pulse group secreted more insulin at basal ( time zero ) and stimulatory concentrations of glucose ( 16.7  mm ) between 5 and 60  min following stimulation ( figure  1e ) .
this effect persisted even after glucose concentrations were returned to 2.8  mm . in parallel , the phosphorylation of camkii was higher in the glucose pulse group at both glucose concentrations , and remained elevated even after the glucose concentration was reduced to 2.8  mm ( figure  1f ) .
thus , min6  - cells had acquired metabolic memory of prior exposure to glucose pulses , which they retained through a 24-h  consolidation period .
it was important to determine if metabolic memory is confined to min6 insulinoma cells , or if it is also a property of primary human  - cells .
therefore , we cultured human islets from multiple non - diabetic deceased organ donors ( see table  1 for donor information ) and exposed them to pulses of 30  mm glucose in the same paradigm as described above .
pulse treatment increased insulin secretion at basal ( 2.8  mm ) and stimulatory ( 16.8  mm ) glucose concentrations ( figure  2a ) . as was the case in min6  - cells ,
the pulse group showed increased camkii phosphorylation at both basal and stimulatory glucose concentrations ( figure  2b ) .
next , we determined the human islet response to a glucose challenge in the islet perifusion assay .
islets from the glucose pulse group showed both increased first and second phase insulin secretion compared with islets from the control group ( figure  2c and d ) .
as before , these effects were abolished by the camkii inhibitor kn93 ( figure  2a  d ) .
we also tested metabolic memory of  - cells using isolated mouse pancreatic islets , with similar outcome ( extended data figure  1 )
. we also found , using live cell calcium imaging of mouse islets , that the increases in cytoplasmic calcium levels following acute glucose stimulation , representing the penultimate step in insulin secretion , are significantly higher in the pulse group compared with controls ( extended data figure  1c ) . to investigate the molecular mechanisms by which the glucose pulse paradigm induces memory in  - cells , we evaluated the expression of essential mediators of the insulin secretory response .
the glucose pulse paradigm induced expression of glucokinase ( gck ) , the main glucose sensor of  - cells ( figure  3a ) , the voltage - gated ca channel ( cav1.2 ) ( figure  3b ) , snap25 , an essential component of the exocytotic machinery ( figure  3c ) , and mafa , a key transcription factor of the insulin gene and the mature  - cell phenotype in general ( figure  3d ) .
all the effects of the pulsed glucose paradigm on protein levels were reduced or abolished by treatment with kn93 during the high glucose exposure only . finally , we asked if metabolic memory of  - cells is dependent on glucose metabolism itself , or if this memory can be produced by membrane depolarization alone . to this end , we treated human islets with pulses of 30  mm kcl , which causes membrane depolarization , opening of voltage - gated ca channels , and insulin secretion without a prior increase in intracellular atp levels .
as shown in figure  3e , depolarization by potassium chloride during the acquisition period produced similar , if somewhat lower , increases in pcamkii and psynapsin levels 24  h  after the last kcl pulse .
thus , increases in intracellular calcium elicited by membrane depolarization are sufficient to activate camkii and produce metabolic memory .
total camkii protein levels were not affected by any of the treatments used ( data not shown ) .
glucose - stimulated insulin secretion is a complex process that translates glycolytic flux and elevated atp production to increased cytoplasmic ca levels and finally fusion of insulin granules with the plasma membrane .
this process is accompanied by increased phosphorylation of camkii ( extended data figure  2 ) , a process now shown to be part of the establishment of metabolic memory in  - cells .
cerasi and colleagues had previously reported time - dependent potentiation of insulin secretion , in that exposure of rat islets to high glucose ( 27.7  mm ) up to 60  min prior to the test stimulation with glucose increased subsequent insulin secretion .
however , these studies focused on analyzing acute effects of high glucose pre - treatment , and did not yet investigate the molecular mechanism underlying the phenomenon . here ,
we demonstrate that human and mouse pancreatic  - cells are able to acquire , consolidate , and retrieve information , induced by high glucose exposure , and determined that this ability is dependent on camkii . in conclusion
, we have shown here that , like neurons , human and mouse  - cells are able to acquire , store , and retrieve information in a process similar to neuronal ltp .
this process , presented schematically in figure  4 , is dependent on the activation of camkii , as is the case for neuronal memory .
these findings provide further evidence that the similarity of neurons and  - cells on the transcriptome and epigenome level is not accidental , but an important aspect of the biology of these embryologically distinct cell types
. metabolic memory of  - cells likely represents a useful evolutionary adaptation to variation in food availability . as periods of food abundance in nature
can vary dramatically in length , metabolic memory of repeated carbohydrate loads in  - cells ensures higher insulin secretion and thus more efficient uptake of excess glucose into skeletal muscle and adipose tissue for long - term storage , contributing to the adaptation to periods of starvation .
given the pivotal role of altered insulin secretion in metabolic disorders , future work will need to establish to what extent metabolic memory of pancreatic  - cells contributes to the pathophysiology of these diseases .
 ca2+/calmodulin - dependent protein kinase ii ( camkii ) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators . 
 therefore , we hypothesized that pancreatic  - cells acquire and store the information contained in calcium pulses as a form of  metabolic memory  , just as neurons store cognitive information . to test this hypothesis , we developed a novel paradigm of pulsed exposure of  - cells to intervals of high glucose , followed by a 24-h  consolidation period to eliminate any acute metabolic effects . 
 strikingly ,  - cells exposed to this high - glucose pulse paradigm exhibited significantly stronger insulin secretion . 
 this metabolic memory was entirely dependent on camkii . 
 metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and ca2 + -dependent vesicle secretion , and by elevated levels of the key  - cell transcription factor mafa . in summary , like neurons , 
 human and mouse  - cells are able to acquire and retrieve information . 
Introduction
Materials and methods
Results and discussion