ins-1 cells ( passage 545 ) were cultured in rpmi1640 medium ( sigma ) containing 11.1 mmol / l d - glucose supplemented with 10% heat - inactivated fbs , 10 mmol / l hepes , 100 units / ml penicillin , 100 mg / ml streptomycin , 1 mmol / l sodium pyruvate , and 50 mol / l -mercaptoethanol at 37c in a humidified incubator containing 5% co2 .
cells were grown in 12-well plates at a density of 5  10 per well for electrophysiology and on 12-mm poly - l - lysine  coated coverslips for immunocytochemistry .
pancreatic -cells were isolated from the islets of langerhans of sprague - dawley rats weighing 200300 g. animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) .
all procedures were approved by the institute of laboratory animal resources at seoul national university .
islets were isolated after digesting the pancreas by injecting 1 mg / ml collagenase dissolved in hank 's balanced salt saline ( hbss , sigma ) solution into pancreatic ducts .
collected islets were dispersed mechanically into single cells in ca - free krebs - ringer bicarbonate buffer ( krbb , sigma ) and then plated onto poly - l - lysine  coated coverslips and cultured for 45 days in rpmi1640 medium containing 11.1 mmol / l d - glucose , 100 units / ml penicillin , and 100 mg / ml streptomycin .
we confirmed by poly(adp - ribose ) polymerase cleavage assay that cell viability was not affected after glucose deprivation for 2 h. katp channel currents were monitored at room temperature using a standard whole - cell or inside - out patch - clamp technique with an epc-10 amplifier and pulse software ( version 8.67 ; heka electronik ) and analyzed using igor software .
the cell - attached patch technique was used to measure unitary katp currents with an axopatch 200b amplifier and analyzed with pclamp software ( axon instruments ) .
patch electrodes were pulled from borosilicate glass capillaries to obtain a resistance between 2 and 5 mol / l. the internal solution for whole - cell experiments contained ( in millimoles per liter ) 30 kcl , 110 k - aspartate , 2.6 cacl2 , 10 hepes ( ph 7.2 with koh ) , 0.5 egta , and 5 edta .
the bath solution for whole - cell and cell - attached patch recordings contained ( in millimoles per liter ) 137 nacl , 5.6 kcl , 10 hepes ( ph 7.4 with naoh ) , 0.5 mgcl2 , and 1.8 cacl2 .
compound c ( calbiochem ) and 5-aminoimidazole-4-carboxamide ribonucleoside ( aicar , calbiochem ) were added during pretreatment as indicated in the text and included during current recordings . for inside - out and cell - attached patch recordings ,
pipettes were filled with a solution containing ( in millimoles per liter ) 140 kcl , 2.6 cacl2 , 1 mgcl2 , and 10 hepes ( ph 7.2 with koh ) ; upon patch excision in inside - out experiments , bath solutions were changed to a solution containing ( in millimoles per liter ) 107 kcl , 1 cacl2 , 2 mgcl2 , 10 hepes , and 11 egta ( ph 7.2 with koh ) .
cells were fixed with 4% paraformaldehyde in pbs for 15 min . for katp channel staining
, cells were permeabilized with 0.25% triton x-100 in pbs for 10 min , blocked with 2% donkey serum in pbs for 30 min at room temperature , and incubated with rabbit anti - kir6.2 ( 1:50 , santa cruz biotechnology ) and goat anti - sur1 ( 1:50 , santa cruz biotechnology ) and rabbit anti kir2.1 ( 1:50 , santa cruz biotechnology ) antibodies overnight at 4c .
the subcellular localizations of kir6.2 were assessed using alexa488-conjugated anti - rabbit igg antibody ( 488-nm excitation , 1:100 , invitrogen ) and cy5-conjugated anti - goat igg ( 633-nm excitation , 1:100 , jackson immunoresearch ) antibodies , respectively .
images were acquired by confocal microscopy ( leica , germany ; fluoview 1000 , olympus ) using a 63 oil - immersion objective .
the same instrument settings were used for each experiment , and all experiments were repeated at least three times . because we did not identify -cells using insulin antibody , we could not entirely exclude the possibility that -cells or -cells were included in our observation .
the surface and total expression levels of kir6.2 were determined by western blot analysis using an antibody to kir6.2 ( 1:1,000 , santa cruz biotechnology ) .
the surface protein fractions of ins-1 cells were assessed using surface biotinylation ez - link sulfo - nhs - ss - biotin kits ( pierce ) . to verify consistent amounts of surface proteins , the membrane probed by kir6.2 primary antibody was washed for 2 h and examined by immunoblot using an antibody to kir2.1 ( 1:1,000 , santa cruz biotechnology ) .
total protein was prepared by incubating cells with ripa buffer , and proteins were resolved by 12% sds - page .
insulin secretion from rat pancreatic islets was measured by enzyme immunoassay ( elisa kits , mercodia ) .
changes in insulin secretion in response to glucose deprivation were monitored using the batch incubation method by collecting samples at 30-min intervals .
all statistical analyses were performed using anova , and p < 0.05 was considered to be significant .
ins-1 cells ( passage 545 ) were cultured in rpmi1640 medium ( sigma ) containing 11.1 mmol / l d - glucose supplemented with 10% heat - inactivated fbs , 10 mmol / l hepes , 100 units / ml penicillin , 100 mg / ml streptomycin , 1 mmol / l sodium pyruvate , and 50 mol / l -mercaptoethanol at 37c in a humidified incubator containing 5% co2 .
cells were grown in 12-well plates at a density of 5  10 per well for electrophysiology and on 12-mm poly - l - lysine  coated coverslips for immunocytochemistry .
pancreatic -cells were isolated from the islets of langerhans of sprague - dawley rats weighing 200300 g. animals were anesthetized with pentobarbital sodium ( 50 mg / kg , i.p . ) .
all procedures were approved by the institute of laboratory animal resources at seoul national university .
islets were isolated after digesting the pancreas by injecting 1 mg / ml collagenase dissolved in hank 's balanced salt saline ( hbss , sigma ) solution into pancreatic ducts .
collected islets were dispersed mechanically into single cells in ca - free krebs - ringer bicarbonate buffer ( krbb , sigma ) and then plated onto poly - l - lysine  coated coverslips and cultured for 45 days in rpmi1640 medium containing 11.1 mmol / l d - glucose , 100 units / ml penicillin , and 100 mg / ml streptomycin .
we confirmed by poly(adp - ribose ) polymerase cleavage assay that cell viability was not affected after glucose deprivation for 2 h.
katp channel currents were monitored at room temperature using a standard whole - cell or inside - out patch - clamp technique with an epc-10 amplifier and pulse software ( version 8.67 ; heka electronik ) and analyzed using igor software .
the cell - attached patch technique was used to measure unitary katp currents with an axopatch 200b amplifier and analyzed with pclamp software ( axon instruments ) .
patch electrodes were pulled from borosilicate glass capillaries to obtain a resistance between 2 and 5 mol / l. the internal solution for whole - cell experiments contained ( in millimoles per liter ) 30 kcl , 110 k - aspartate , 2.6 cacl2 , 10 hepes ( ph 7.2 with koh ) , 0.5 egta , and 5 edta .
the bath solution for whole - cell and cell - attached patch recordings contained ( in millimoles per liter ) 137 nacl , 5.6 kcl , 10 hepes ( ph 7.4 with naoh ) , 0.5 mgcl2 , and 1.8 cacl2 .
compound c ( calbiochem ) and 5-aminoimidazole-4-carboxamide ribonucleoside ( aicar , calbiochem ) were added during pretreatment as indicated in the text and included during current recordings . for inside - out and
cell - attached patch recordings , pipettes were filled with a solution containing ( in millimoles per liter ) 140 kcl , 2.6 cacl2 , 1 mgcl2 , and 10 hepes ( ph 7.2 with koh ) ; upon patch excision in inside - out experiments , bath solutions were changed to a solution containing ( in millimoles per liter ) 107 kcl , 1 cacl2 , 2 mgcl2 , 10 hepes , and 11 egta ( ph 7.2 with koh ) .
cells were fixed with 4% paraformaldehyde in pbs for 15 min . for katp channel staining , cells
were permeabilized with 0.25% triton x-100 in pbs for 10 min , blocked with 2% donkey serum in pbs for 30 min at room temperature , and incubated with rabbit anti - kir6.2 ( 1:50 , santa cruz biotechnology ) and goat anti - sur1 ( 1:50 , santa cruz biotechnology ) and rabbit anti kir2.1 ( 1:50 , santa cruz biotechnology ) antibodies overnight at 4c .
the subcellular localizations of kir6.2 were assessed using alexa488-conjugated anti - rabbit igg antibody ( 488-nm excitation , 1:100 , invitrogen ) and cy5-conjugated anti - goat igg ( 633-nm excitation , 1:100 , jackson immunoresearch ) antibodies , respectively .
images were acquired by confocal microscopy ( leica , germany ; fluoview 1000 , olympus ) using a 63 oil - immersion objective .
the same instrument settings were used for each experiment , and all experiments were repeated at least three times . because we did not identify -cells using insulin antibody , we could not entirely exclude the possibility that -cells or -cells were included in our observation .
the surface and total expression levels of kir6.2 were determined by western blot analysis using an antibody to kir6.2 ( 1:1,000 , santa cruz biotechnology ) .
the surface protein fractions of ins-1 cells were assessed using surface biotinylation ez - link sulfo - nhs - ss - biotin kits ( pierce ) . to verify consistent amounts of surface proteins , the membrane probed by kir6.2 primary antibody was washed for 2 h and examined by immunoblot using an antibody to kir2.1 ( 1:1,000 , santa cruz biotechnology ) .
total protein was prepared by incubating cells with ripa buffer , and proteins were resolved by 12% sds - page .
insulin secretion from rat pancreatic islets was measured by enzyme immunoassay ( elisa kits , mercodia ) .
changes in insulin secretion in response to glucose deprivation were monitored using the batch incubation method by collecting samples at 30-min intervals .
all statistical analyses were performed using anova , and p < 0.05 was considered to be significant .
to investigate the roles of energy - dependent signaling mechanisms in katp channel regulation , we pretreated ins-1 cells with different concentrations of glucose ( 0
mmol / l for glucose - deprived [ gd ] , 3 mmol / l for low glucose [ 3 g ] , 11.1 mmol / l for control [ 11 g ] ) for 2 h at room temperature before katp currents were recorded .
1a . to induce the maximal activation of katp channels , we dialyzed the cells with atp - free internal solutions and monitored changes in cell conductance in response to the washout of intracellular atp by applying hyperpolarizing pulses to 130 mv , from a holding potential of 70 mv every 10 s ( fig .
we confirmed that the increases in currents under these conditions were because of katp channel activation by testing the effect of glibenclamide , a selective katp channel blocker ( data not shown ) .
for reliable measurements of maximum current amplitude , the katp current rundown was minimized by adding 5 mmol / l edta to the internal solutions ( 19 ) . upon patch break - in ,
several minutes passed until the whole - cell conductance increased rapidly and reached the maximum level .
this delay was longer in cells pretreated with 11 g than that in cells pretreated with 3 g or the gd solution .
this observation was expected because the initial atp concentrations would be higher in 11g - treated cells , so that atp washout would take longer .
however , it was surprising to find that the amplitudes of katp currents at their maximum activation were much smaller in 11g - treated cells than in 3g- or gd - treated cells ( fig .
the maximum katp conductance ( gmax ) , calculated by measuring the difference in current amplitudes between 130 and 70 mv , was normalized to the cell capacitance ( cm ) .
ns / pf ) was similar to that in 3g - treated cells ( 2.28  0.12 ns / pf ) , whereas that in 11g - treated cells ( 0.33  0.05 ns / pf ) was significantly less than that in gd- or 3g - treated cells ( p
no significant difference in cm was observed among 11 g , 3 g , and gd - treated cells ( 12.01  0.28 pf ; n = 130 ) .
. a : schematic diagram of the experimental protocols used for treating cells before current recording .
11 g , 3 g , and gd represent 11.1 mmol / l , 3 mmol / l , and 0 mmol / l glucose , respectively .
b : representative recordings of katp whole - cell currents in 11g- , 3g- , and gd - treated ins-1 cells .
whole - cell current traces were obtained by hyperpolarizing pulses to 130 mv ( 500 ms ) from a holding potential of 70 mv every 10 s. c : mean conductance normalized to cell capacitance ( ns / pf ) measured at peak activation from 11g- , 3g- , and gd - treated cells ( n = 8 , 11 , and 12 , respectively ) .
d : single katp channel currents recorded at 60 mv from an inside - out patch obtained from 11g- ( upper panel ) and gd - treated ins-1 cells ( lower panel ) exposed to 0.25 mmol / l diazoxide in the presence of 1 mol / l mgatp .
e : upper panel : mean katp channel activity ( npo ) in 11g- and gd - treated cells .
mean values for npo ; 0.16  0.04 ( n = 28 ) in 11 g and 0.86  0.12 ( n = 28 ) in gd , 0.05  0.02 ( n = 16 ) in 11 g and 0.25  0.06 ( n = 28 ) in gd , 0.04  0.01 ( n = 8) in 11 g and 0.15  0.04 ( n = 10 ) in gd when the cells were exposed to 0.25 mmol / l diazoxide in the presence of 1 mol / l , 10 mol / l , and 100 mol / l mgatp , respectively .
lower panel : maximum number of channels opened at the same time ( n ' ) within the excised patch pipette .
statistical significance was evaluated by an unpaired t test and one - way anova . * * * p < 0.001 .
it may be argued whether the lower gmax in 11g - treated cells was because of insufficient washout of cellular factors that could suppress katp channels .
to exclude this possibility , we used inside - out excised patch recordings , yet significant differences in katp channel activities were still observed between 11g- and gd - treated cells . the mean value for npo obtained in the presence of diazoxide ( 0.25 mmol / l ) and atp in gd - treated cells was about five times larger than that in 11g - treated cells ( fig .
furthermore , the maximum number of channels opened at the same time in a patch membrane ( n ' ) was significantly more in gd - treated cells compared with 11g - treated cells . the differences between 11g- and gd - treated cells were unaffected by varying atp concentrations ( fig
these results support the hypothesis that some energy - dependent signaling mechanism(s ) activated during the pretreatment in 3 g or gd was required to activate katp conductance to its full capacity .
there was no evidence that the atp sensitivity and gating kinetics ( mean open time ) of the katp channels were affected by glucose deprivation ( supplementary figs . s1 and s2 ) .
we next investigated which signaling mechanism is responsible for the gd - induced increases in katp channels .
the activation of ampk by glucose deprivation has been reported in insulin - secreting cell lines ( 18 ) . to confirm that ampk was activated in ins-1 cells by glucose deprivation or low glucose ( 3
mmol / l ) , western blot analysis was performed using a phosphorylation - specific antibody .
the levels of the phosphorylated ampk ( pthr ) increased similarly for cells in 0 or 3 mmol / l glucose solutions , reaching maximum levels after 2 h at room temperature and 30 min at 37c , whereas the total ampk levels were unaffected ( fig .
we tested the involvement of ampk in gd - induced increases in katp channel activities using compound c ( an ampk inhibitor ) ( 20 ) , ampk sirna transfection , and aicar ( an ampk activator ) ( 21 ) .
mol / l ) for 30 min before current recording in glucose - free solution ( gd + cc ; fig .
2b , upper panel ) , and gmax ( 0.22  0.04 ns / pf ; n = 11 ) became similar to the background conductance measured in the presence of glibenclamide ( 0.12  0.03 ns / pf ) .
we confirmed that the katp channel activity recorded by inside - out patches was not affected by compound c applied to the internal side of the membrane ( supplementary fig .
s3b ) , but katp current activation in gd - treated cells in whole - cell recordings was abolished by adding compound c to internal solutions ( supplementary fig .
these results indicated that compound c inhibited katp channels not directly but by inhibiting intracellular mechanisms and that this inhibitory effect appeared in a relatively short period .
furthermore , katp current activation in 11g - treated cells increased significantly when aicar ( 0.5 mmol / l ) was added 20 min before current recording ( fig . 2b , middle panel ) ,
the application of aicar to gd - treated cells did not induce further increases in gmax .
the effects of compound c and aicar on katp channel activities observed in inside - out recordings ( fig .
the finding that the effect of glucose deprivation on katp channels was inhibited by compound c and mimicked by aicar indicated that ampk was responsible for the gd - induced increases in katp conductance .
we confirmed that the effects of gd , compound c , and aicar on katp conductance were similarly observed in pancreatic -cells ( supplementary fig .
a : the levels of phosphorylated ampk ( upper panel ) and total ampk ( lower panel ) measured by western blot assay from cell lysates of ins-1 cells pretreated with 3 g or gd solution for the times indicated at room temperature ( 25c , left panel ) and 37c ( right panel ) .
b : katp whole - cell currents recorded in cells pretreated with compound c ( 10
mol / l ) in glucose - free solution ( gd + cc , upper panel ) and in cells pretreated with aicar ( 0.5 mmol / l ) in 11.1 mmol / l glucose solution ( 11 g + aicar , middle panel ) .
lower panel : normalized mean conductance ( ns / pf ) obtained in gd + cc ( 0.22  0.04 ; n = 11 ) , 11 g + aicar ( 2.10  0.43 ; n = 6 ) , and gd + aicar ( 2.44  0.40 ; n = 6 ) are compared with those of gd and 11 g shown in fig . 1c .
c : single katp channel currents recorded at 60 mv in an inside - out patch obtained from gd + cc ( upper panel ) and 11 g + aicar treated ins-1 cells ( middle panel ) . mean values for npo obtained in gd + cc ( 0.06  0.02 ; n = 13 ) and 11 g + aicar ( 0.20  0.06 ; n = 7 ) are compared with those of gd and 11 g shown in fig .
d : whole - cell current traces obtained from ins-1 cells transfected with scr or siampk and pretreated in zero glucose for 2 h. normalized gmax ( in ns / pf ) ; 0.39  0.17 ( n = 7 ) in siampk - transfected gd cells and 3.26  0.52 ( n = 7 ) in scr - transfected gd cells .
the gmax values in two gd + siampk cells that display exceptionally large currents are indicated separately by open circles above the bar .
statistical significance was evaluated by unpaired t test and one - way anova . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 .
the involvement of ampk in katp channel regulation was further confirmed in ins-1 cells transfected with both 1- and 2-ampk sirna ( siampk ) ( research design and methods are described in the online appendix ) .
the total expression of the -subunit of ampk in cells transfected with siampk was significantly lower than that in cells transfected with an equal concentration of scrambled sirna ( scr ; supplementary fig .
green fluorescence protein was cotransfected , and green fluorescence protein - positive cells were selected for current recordings .
transfection of scr did not appear to affect the katp current in gd - treated cells ( fig .
2d , lower panel ) , and the normalized gmax was similar to that in untransfected cells ( fig .
1 ) . in contrast , the katp current activation in cells transfected with siampk was negligible in seven of nine cells ( fig . 2d , upper panel ) .
in two cells , katp current activation was well observed , probably because of the variation in ampk knockdown levels among cells .
the gmax values in these cells were not included in the calculation of the mean value and are shown separately with open circles above the bar ( fig .
, higher katp channel activity can be caused by increasing the number of functional channels ( n ) or by increasing the open probability ( po ) .
because the measurement of gmax or npo can not distinguish these two possibilities ( further discussion in the online appendix ) , we examined changes in the surface expression levels of kir6.2 using surface biotinylation / streptavidin purification and subsequent western blot analysis in ins-1 cells . for the loading control , we probed the same blots with an antibody to kir2.1 , another inward - rectifying k channel .
we found that the kir6.2 surface levels were low in 11g- or 6g - treated cells ( supplementary fig .
s5b ) but increased similarly in 3g- and gd - treated cells ( duration of pretreatment : 2 h ; fig .
the increase in kir6.2 surface level was dependent on the duration of glucose deprivation up to 2 h ( fig .
3a ) , which was consistent with the effect of glucose deprivation on ampk activation ( fig .
in contrast , the increase in surface kir6.2 by glucose deprivation was abolished by compound c ( duration of compound c application : 20 min in fig .
s5b ) . increased kir6.2 surface expression was not induced by inducing hyperpolarization using a katp channel opener ( diazoxide ; supplementary fig .
s5b ) but induced by the addition of aicar to 11 g solution ( fig .
kir2.1 surface levels remained unchanged by these treatments , indicating that the above effects were specific to kir6.2 .
we also verified that the total kir6.2 levels detected by directly immunoblotting the cell lysates did not change under these conditions , indicating that the ampk - induced signaling activated during 3 g or gd treatments regulated kir6.2 trafficking to increase the kir6.2 surface levels without changes in gene expression .
a : ins-1 cells were incubated with gd medium for the times indicated before surface labeling with a biotin probe .
the surface ( s ) or total ( t ) expression level of kir6.2 was assessed by western blot analysis as described in the online appendix .
kir2.1 , a surface channel that does not react on glucose deprivation , was used as a negative control .
b : the cells were maintained with 11 g , 3 g , or gd medium in the presence or absence of 40 mol / l compound c for 2 h before surface labeling with a biotin probe .
the surface expression level of kir6.2 was quantified by densitometry and expressed as an arbitrary value to that of the cells incubated with gd medium , which was set to 1 .
c : the cells were treated with 0.5 mmol / l aicar - containing 11 g medium for the times indicated before surface labeling with a biotin probe .
the surface expression level of kir6.2 was quantified by using the same method in b. statistical significance was determined by anova . *
. to observe changes in channel protein distributions , we used confocal microscopy with fluorescent - labeled antibodies to kir6.2 ( green ) in pancreatic -cells .
the nucleus and plasma membrane were located using dii ( red ) and dapi ( blue ) , respectively . in 11g - treated cells , punctuate structures were observed scattered in the cytosol ( fig .
4a ) , whereas in gd - treated cells , dominant fluorescence signals were observed at the cell periphery ( fig .
the fluorescence intensity profiles measured along a line drawn across the cell membrane excluding the nucleus also confirmed that the areas of high intensity were scattered randomly in 11g - treated cells and at the cell periphery in gd - treated cells .
the kir6.2 signals at the cell periphery exhibited broader signal intensity than the red fluorescence signals emitted by plasma membrane marker dii , suggesting that the amount kir6.2 subunits underneath the membrane , as well as integrated at the surface membrane , were higher in gd - treated cells .
when compound c was added to the glucose - deprived medium , the effects of glucose deprivation on the distribution of kir6.2 subunits were abolished ( fig .
when aicar was added to the 11 g solution , the distribution pattern and intensity profile of kir6.2 subunits were similar to those of gd - treated cells ( fig .
these results indicate that ampk activation increases katp channel surface expression by regulating the trafficking of katp channel proteins .
we confirmed that sur1 distribution was changed by each treatment similarly to those observed for kir6.2 subunits ( supplementary fig .
d : confocal fluorescent images obtained from pancreatic -cells immunolabeled with kir6.2 antibody ( green , second panel ) in 11 g ( a ) , gd ( b ) , gd + cc ( c ) , and 11 g + aicar ( d ) .
both compound c ( 10 mol / l ) and aicar ( 0.5 mmol / l ) were pretreated for 1 h before fixation .
nucleus was stained with dapi ( blue , first panel ) , and plasma membrane was stained with dii ( red , third panel ) .
cells were incubated with these dyes ( dapi , 405-nm excitation , 1:5,000 , molecular probes ; dii , 543-nm excitation , 1:1,000 , sigma ) for 5 min immediately before mounting .
overlay of the fluorescence images showing kir6.2 , dapi , and dii ( fourth panel ) .
the intensity profiles of kir6.2 ( green ) and dii ( red ) fluorescence measured along a yellow line drawn across the cell membrane excluding the nucleus ( fifth panel ) .
( a high - quality color digital representation of this figure is available in the online issue . ) to test the functional significance of the ampk - dependent modulation of katp channels , we examined whether compound c and aicar cause decreases in insulin secretion in response to glucose deprivation ( fig .
the insulin secretion measured at 11.1 mmol / l glucose was higher in the presence of compound c , but lower in the presence of aicar when compared with the control .
after glucose deprivation , the release of insulin decreased gradually to the minimum level within 1 h for the control ; this response was inhibited significantly by compound c , whereas pretreatment with aicar facilitated the decrease in insulin secretion in response to glucose deprivation .
effects of compound c and aicar on decreased insulin secretion because of glucose deprivation . a : experimental protocol showing of how islets were incubated in 11.1 mmol
/ l glucose for 1 h before removing the glucose ( time set as 0 min ) .
the samples for insulin measurements were taken from each independent batch at 30 , 0 , 30 , 60 , 90 , and 120 min ( arrows ) in the krb buffer .
b : time - dependent changes in the percentage of islet insulin content secreted during acute glucose deprivation .
insulin secretion as a percentage of total insulin content ( secreted / total ) was calculated .
control ( circles ) ; in the presence of compound c ( triangles ) ; in the presence of aicar ( squares ) . for each data point
* p < 0.05 , * * p < 0.01 , * * * p < 0.001 compared with the control .
to investigate the roles of energy - dependent signaling mechanisms in katp channel regulation , we pretreated ins-1 cells with different concentrations of glucose ( 0
mmol / l for glucose - deprived [ gd ] , 3 mmol / l for low glucose [ 3 g ] , 11.1 mmol / l for control [ 11 g ] ) for 2 h at room temperature before katp currents were recorded .
1a . to induce the maximal activation of katp channels , we dialyzed the cells with atp - free internal solutions and monitored changes in cell conductance in response to the washout of intracellular atp by applying hyperpolarizing pulses to 130 mv , from a holding potential of 70 mv every 10 s ( fig .
we confirmed that the increases in currents under these conditions were because of katp channel activation by testing the effect of glibenclamide , a selective katp channel blocker ( data not shown ) .
for reliable measurements of maximum current amplitude , the katp current rundown was minimized by adding 5 mmol / l edta to the internal solutions ( 19 ) . upon patch break - in ,
several minutes passed until the whole - cell conductance increased rapidly and reached the maximum level .
this delay was longer in cells pretreated with 11 g than that in cells pretreated with 3 g or the gd solution .
this observation was expected because the initial atp concentrations would be higher in 11g - treated cells , so that atp washout would take longer .
however , it was surprising to find that the amplitudes of katp currents at their maximum activation were much smaller in 11g - treated cells than in 3g- or gd - treated cells ( fig .
the maximum katp conductance ( gmax ) , calculated by measuring the difference in current amplitudes between 130 and 70 mv , was normalized to the cell capacitance ( cm ) .
ns / pf ) was similar to that in 3g - treated cells ( 2.28  0.12 ns / pf ) , whereas that in 11g - treated cells ( 0.33  0.05 ns / pf ) was significantly less than that in gd- or 3g - treated cells ( p
no significant difference in cm was observed among 11 g , 3 g , and gd - treated cells ( 12.01  0.28 pf ; n = 130 ) .
. a : schematic diagram of the experimental protocols used for treating cells before current recording .
11 g , 3 g , and gd represent 11.1 mmol / l , 3 mmol / l , and 0 mmol / l glucose , respectively .
b : representative recordings of katp whole - cell currents in 11g- , 3g- , and gd - treated ins-1 cells .
whole - cell current traces were obtained by hyperpolarizing pulses to 130 mv ( 500 ms ) from a holding potential of 70 mv every 10 s. c : mean conductance normalized to cell capacitance ( ns / pf ) measured at peak activation from 11g- , 3g- , and gd - treated cells ( n = 8 , 11 , and 12 , respectively ) .
d : single katp channel currents recorded at 60 mv from an inside - out patch obtained from 11g- ( upper panel ) and gd - treated ins-1 cells ( lower panel ) exposed to 0.25 mmol / l diazoxide in the presence of 1 mol / l mgatp .
e : upper panel : mean katp channel activity ( npo ) in 11g- and gd - treated cells .
mean values for npo ; 0.16  0.04 ( n = 28 ) in 11 g and 0.86  0.12 ( n = 28 ) in gd , 0.05  0.02 ( n = 16 ) in 11 g and 0.25  0.06 ( n = 28 ) in gd , 0.04  0.01 ( n = 8) in 11 g and 0.15  0.04 ( n = 10 ) in gd when the cells were exposed to 0.25 mmol / l diazoxide in the presence of 1 mol / l , 10 mol / l , and 100 mol / l mgatp , respectively .
lower panel : maximum number of channels opened at the same time ( n ' ) within the excised patch pipette .
statistical significance was evaluated by an unpaired t test and one - way anova . * * * p < 0.001 .
it may be argued whether the lower gmax in 11g - treated cells was because of insufficient washout of cellular factors that could suppress katp channels .
to exclude this possibility , we used inside - out excised patch recordings , yet significant differences in katp channel activities were still observed between 11g- and gd - treated cells . the mean value for npo obtained in the presence of diazoxide ( 0.25 mmol / l ) and atp in gd - treated cells was about five times larger than that in 11g - treated cells ( fig .
furthermore , the maximum number of channels opened at the same time in a patch membrane ( n ' ) was significantly more in gd - treated cells compared with 11g - treated cells . the differences between 11g- and gd - treated cells were unaffected by varying atp concentrations ( fig
these results support the hypothesis that some energy - dependent signaling mechanism(s ) activated during the pretreatment in 3 g or gd was required to activate katp conductance to its full capacity .
there was no evidence that the atp sensitivity and gating kinetics ( mean open time ) of the katp channels were affected by glucose deprivation ( supplementary figs . s1 and s2 ) .
we next investigated which signaling mechanism is responsible for the gd - induced increases in katp channels .
the activation of ampk by glucose deprivation has been reported in insulin - secreting cell lines ( 18 ) . to confirm that ampk was activated in ins-1 cells by glucose deprivation or low glucose ( 3 mmol / l ) , western blot analysis
the levels of the phosphorylated ampk ( pthr ) increased similarly for cells in 0 or 3 mmol / l glucose solutions , reaching maximum levels after 2 h at room temperature and 30 min at 37c , whereas the total ampk levels were unaffected ( fig .
we tested the involvement of ampk in gd - induced increases in katp channel activities using compound c ( an ampk inhibitor ) ( 20 ) , ampk sirna transfection , and aicar ( an ampk activator ) ( 21 ) .
mol / l ) for 30 min before current recording in glucose - free solution ( gd + cc ; fig .
2b , upper panel ) , and gmax ( 0.22  0.04 ns / pf ; n = 11 ) became similar to the background conductance measured in the presence of glibenclamide ( 0.12  0.03 ns / pf ) .
we confirmed that the katp channel activity recorded by inside - out patches was not affected by compound c applied to the internal side of the membrane ( supplementary fig .
s3b ) , but katp current activation in gd - treated cells in whole - cell recordings was abolished by adding compound c to internal solutions ( supplementary fig .
these results indicated that compound c inhibited katp channels not directly but by inhibiting intracellular mechanisms and that this inhibitory effect appeared in a relatively short period .
furthermore , katp current activation in 11g - treated cells increased significantly when aicar ( 0.5 mmol / l ) was added 20 min before current recording ( fig . 2b , middle panel ) , becoming indistinguishable from katp current activation of gd - treated cells .
the application of aicar to gd - treated cells did not induce further increases in gmax .
the effects of compound c and aicar on katp channel activities observed in inside - out recordings ( fig .
the finding that the effect of glucose deprivation on katp channels was inhibited by compound c and mimicked by aicar indicated that ampk was responsible for the gd - induced increases in katp conductance .
we confirmed that the effects of gd , compound c , and aicar on katp conductance were similarly observed in pancreatic -cells ( supplementary fig .
a : the levels of phosphorylated ampk ( upper panel ) and total ampk ( lower panel ) measured by western blot assay from cell lysates of ins-1 cells pretreated with 3 g or gd solution for the times indicated at room temperature ( 25c , left panel ) and 37c ( right panel ) .
b : katp whole - cell currents recorded in cells pretreated with compound c ( 10
mol / l ) in glucose - free solution ( gd + cc , upper panel ) and in cells pretreated with aicar ( 0.5 mmol / l ) in 11.1 mmol / l glucose solution ( 11 g + aicar , middle panel ) .
lower panel : normalized mean conductance ( ns / pf ) obtained in gd + cc ( 0.22  0.04 ; n = 11 ) , 11 g + aicar ( 2.10  0.43 ; n = 6 ) , and gd + aicar ( 2.44  0.40 ; n = 6 ) are compared with those of gd and 11 g shown in fig .
c : single katp channel currents recorded at 60 mv in an inside - out patch obtained from gd + cc ( upper panel ) and 11 g + aicar treated ins-1 cells ( middle panel ) . mean values for npo obtained in gd + cc ( 0.06  0.02 ; n = 13 ) and 11 g + aicar ( 0.20  0.06 ; n = 7 ) are compared with those of gd and 11 g shown in fig .
d : whole - cell current traces obtained from ins-1 cells transfected with scr or siampk and pretreated in zero glucose for 2 h. normalized gmax ( in ns / pf ) ; 0.39  0.17 ( n = 7 ) in siampk - transfected gd cells and 3.26  0.52 ( n = 7 ) in scr - transfected gd cells .
the gmax values in two gd + siampk cells that display exceptionally large currents are indicated separately by open circles above the bar .
statistical significance was evaluated by unpaired t test and one - way anova . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 .
the involvement of ampk in katp channel regulation was further confirmed in ins-1 cells transfected with both 1- and 2-ampk sirna ( siampk ) ( research design and methods are described in the online appendix ) .
the total expression of the -subunit of ampk in cells transfected with siampk was significantly lower than that in cells transfected with an equal concentration of scrambled sirna ( scr ; supplementary fig .
, green fluorescence protein was cotransfected , and green fluorescence protein - positive cells were selected for current recordings .
transfection of scr did not appear to affect the katp current in gd - treated cells ( fig .
2d , lower panel ) , and the normalized gmax was similar to that in untransfected cells ( fig .
1 ) . in contrast , the katp current activation in cells transfected with siampk was negligible in seven of nine cells ( fig . 2d , upper panel ) .
in two cells , katp current activation was well observed , probably because of the variation in ampk knockdown levels among cells .
the gmax values in these cells were not included in the calculation of the mean value and are shown separately with open circles above the bar ( fig .
in principle , higher katp channel activity can be caused by increasing the number of functional channels ( n ) or by increasing the open probability ( po ) .
because the measurement of gmax or npo can not distinguish these two possibilities ( further discussion in the online appendix ) , we examined changes in the surface expression levels of kir6.2 using surface biotinylation / streptavidin purification and subsequent western blot analysis in ins-1 cells . for the loading control , we probed the same blots with an antibody to kir2.1 , another inward - rectifying k channel .
we found that the kir6.2 surface levels were low in 11g- or 6g - treated cells ( supplementary fig .
s5b ) but increased similarly in 3g- and gd - treated cells ( duration of pretreatment : 2 h ; fig .
the increase in kir6.2 surface level was dependent on the duration of glucose deprivation up to 2 h ( fig .
3a ) , which was consistent with the effect of glucose deprivation on ampk activation ( fig .
in contrast , the increase in surface kir6.2 by glucose deprivation was abolished by compound c ( duration of compound c application : 20 min in fig .
s5b ) . increased kir6.2 surface expression was not induced by inducing hyperpolarization using a katp channel opener ( diazoxide ; supplementary fig .
s5b ) but induced by the addition of aicar to 11 g solution ( fig .
kir2.1 surface levels remained unchanged by these treatments , indicating that the above effects were specific to kir6.2 .
we also verified that the total kir6.2 levels detected by directly immunoblotting the cell lysates did not change under these conditions , indicating that the ampk - induced signaling activated during 3 g or gd treatments regulated kir6.2 trafficking to increase the kir6.2 surface levels without changes in gene expression .
a : ins-1 cells were incubated with gd medium for the times indicated before surface labeling with a biotin probe
. the surface ( s ) or total ( t ) expression level of kir6.2 was assessed by western blot analysis as described in the online appendix .
kir2.1 , a surface channel that does not react on glucose deprivation , was used as a negative control .
b : the cells were maintained with 11 g , 3 g , or gd medium in the presence or absence of 40 mol / l compound c for 2 h before surface labeling with a biotin probe .
the surface expression level of kir6.2 was quantified by densitometry and expressed as an arbitrary value to that of the cells incubated with gd medium , which was set to 1 .
c : the cells were treated with 0.5 mmol / l aicar - containing 11 g medium for the times indicated before surface labeling with a biotin probe .
the surface expression level of kir6.2 was quantified by using the same method in b. statistical significance was determined by anova .
. to observe changes in channel protein distributions , we used confocal microscopy with fluorescent - labeled antibodies to kir6.2 ( green ) in pancreatic -cells .
the nucleus and plasma membrane were located using dii ( red ) and dapi ( blue ) , respectively . in 11g - treated cells , punctuate structures were observed scattered in the cytosol ( fig .
4a ) , whereas in gd - treated cells , dominant fluorescence signals were observed at the cell periphery ( fig .
the fluorescence intensity profiles measured along a line drawn across the cell membrane excluding the nucleus also confirmed that the areas of high intensity were scattered randomly in 11g - treated cells and at the cell periphery in gd - treated cells .
the kir6.2 signals at the cell periphery exhibited broader signal intensity than the red fluorescence signals emitted by plasma membrane marker dii , suggesting that the amount kir6.2 subunits underneath the membrane , as well as integrated at the surface membrane , were higher in gd - treated cells .
s6 ) . when compound c was added to the glucose - deprived medium , the effects of glucose deprivation on the distribution of kir6.2 subunits
when aicar was added to the 11 g solution , the distribution pattern and intensity profile of kir6.2 subunits were similar to those of gd - treated cells ( fig .
these results indicate that ampk activation increases katp channel surface expression by regulating the trafficking of katp channel proteins .
we confirmed that sur1 distribution was changed by each treatment similarly to those observed for kir6.2 subunits ( supplementary fig .
d : confocal fluorescent images obtained from pancreatic -cells immunolabeled with kir6.2 antibody ( green , second panel ) in 11 g ( a ) , gd ( b ) , gd + cc ( c ) , and 11 g + aicar ( d ) .
both compound c ( 10 mol / l ) and aicar ( 0.5 mmol / l ) were pretreated for 1 h before fixation . nucleus was stained with dapi ( blue , first panel ) , and plasma membrane was stained with dii ( red , third panel ) .
cells were incubated with these dyes ( dapi , 405-nm excitation , 1:5,000 , molecular probes ; dii , 543-nm excitation , 1:1,000 , sigma ) for 5 min immediately before mounting .
overlay of the fluorescence images showing kir6.2 , dapi , and dii ( fourth panel ) .
the intensity profiles of kir6.2 ( green ) and dii ( red ) fluorescence measured along a yellow line drawn across the cell membrane excluding the nucleus ( fifth panel ) .
( a high - quality color digital representation of this figure is available in the online issue . )
to test the functional significance of the ampk - dependent modulation of katp channels , we examined whether compound c and aicar cause decreases in insulin secretion in response to glucose deprivation ( fig .
the insulin secretion measured at 11.1 mmol / l glucose was higher in the presence of compound c , but lower in the presence of aicar when compared with the control .
after glucose deprivation , the release of insulin decreased gradually to the minimum level within 1 h for the control ; this response was inhibited significantly by compound c , whereas pretreatment with aicar facilitated the decrease in insulin secretion in response to glucose deprivation .
effects of compound c and aicar on decreased insulin secretion because of glucose deprivation . a : experimental protocol showing of how islets were incubated in 11.1 mmol
/ l glucose for 1 h before removing the glucose ( time set as 0 min ) . the samples for insulin measurements were taken from each independent batch at 30 , 0 , 30 , 60 , 90 , and 120 min ( arrows ) in the krb buffer .
b : time - dependent changes in the percentage of islet insulin content secreted during acute glucose deprivation .
insulin secretion as a percentage of total insulin content ( secreted / total ) was calculated .
control ( circles ) ; in the presence of compound c ( triangles ) ; in the presence of aicar ( squares ) . for each data point
* p < 0.05 , * * p < 0.01 , * * * p < 0.001 compared with the control .
activity - dependent and cell signal - dependent trafficking has been reported for many cell surface channels ( e.g. , trpc5 ) ( 22 ) , transporters ( e.g. , glut4 ) ( 23 ) , and receptors ( e.g. , ampa receptors ) ( 24 ) , and these mechanisms play important roles in regulating their activities . the regulatory mechanisms for katp channels have been investigated intensely for many years .
most studies have focused on direct regulation , such as changes in the open probability or the atp sensitivity of the channels ( 25,26 ) , but little is known about how channel numbers at the surface membrane are regulated . to investigate this aspect , we applied current recording , immunocytochemistry , and surface biotinylation techniques .
our results demonstrate that in pancreatic -cells , the surface level of katp channels is dynamically regulated by energy deprivation , resulting in higher katp conductance .
thus , we propose that the activation of katp channels by energy depletion occurs in two steps : ampk - dependent trafficking followed by channel opening via a decrease in atp ( supplementary fig .
the effects of glucose concentration on katp channels in pancreatic -cells have been investigated previously ( 8,27 ) , but the results between studies are not entirely consistent .
( 27 ) reported that an acute reduction of glucose levels induced a rapid increase in the expression of both kir6.2 and sur1 , and this effect was mimicked by the pharmacological activation of ampk .
these findings are consistent with ours , but they interpreted the results to indicate that glucose deprivation upregulates the translation of existing mrnas because they observed no changes in channel distributions or mrnas levels .
( 8) reported that the incubation of -cells in high - glucose solution ( 17 mmol / l ) for 1 h resulted in increased kir6.2 recruitment to the plasma membrane and increased katp currents , and they claimed that pka and ca - dependent signaling is involved in high - glucose 
we do not know the exact reason for this discrepancy , but several important differences exist between experimental conditions ( summarized in supplementary table s1 ) .
differences in the cell model ( mouse vs. rat ; rinm5f vs. ins-1 ; different passage ) , pretreatment protocols ( incubation time and temperature ) , and internal solution composition may account for such discrepancy ( see further details in the online appendix ) .
the seemingly contradictory results may imply that the regulation of the surface density of katp channels involves a complex signaling network and should be considered as a major factor in the regulation of katp channel activities .
although the present study primarily addresses the trafficking regulation of katp channels by ampk , it does not exclude the possibility that ampk - dependent signaling contributes to the regulation of katp gating .
we showed that the effect of ampk signaling on npo measured from excised patches was similar to that on surface kir6.2 levels ( figs . 2 and 3 ) , implying that the changes in npo are mainly attributable to the changes in channel density .
however , ampk inhibition reduced the whole - cell katp conductance more profoundly ( fig .
this difference may imply that ampk signaling in intact cells contributes not only to katp channel trafficking but also to facilitating katp channel gating .
the underlying mechanisms remain to be investigated , but several possibilities can be postulated : ampk may phosphorylate katp channels to increase channel gating or regulate intracellular factors that can facilitate katp channel opening , such as mgadp ( 2830 ) , phosphoinositides ( 26 ) , and long - chain acyl - coas ( 31 ) .
in fact , the inhibition of insulin secretion in a low energy state is a key mechanism in preventing hypoglycemia .
katp channel opening in low atp is generally believed to mediate these responses , but the ic50 for atp blocking measured in excised patches ( 20 mol / l ) is too low to explain katp channel opening at zero or low glucose , in which [ atp]i is at least 0.8 mmol / l ( 30 ) . because the amp : atp ratio varies more sensitively than the adp : atp ratio ( 32 ) , we postulate that the ampk - dependent modulation of katp channels demonstrated in the present study ought to serve as a more sensitive and efficient means of regulating pancreatic -cell excitability and insulin secretion under low - glucose conditions . to test this possibility , we monitored the changes in insulin secretion in response to glucose deprivation and tested the effects of pretreatment with compound c or aicar ( fig .
the results indicated that the reduced insulin secretion by glucose deprivation is significantly attenuated by compound c and facilitated by aicar .
our results appeared to be consistent with the previous studies showing that forced activation of ampk inhibited , whereas compound c increased , glucose- stimulated insulin secretions ( 3335 ) . in those studies ,
however , metabolic effects of ampk were considered to be primarily responsible for regulating insulin secretion .
it has not been studied whether the effects of ampk on katp channel trafficking are involved in this inhibition , but it was shown that ampk decreased the number of docked vesicles containing insulin ( 36,37 ) .
this and the present study indicate that ampk regulates the trafficking of insulin vesicles and kir6.2 in opposing directions , so as to optimize the -cell functions in a low energy state . a similar pattern of concerted action of ampk was reported in epithelia for glut4 and cftr trafficking ( 11 ) . in conclusion ,
katp channel trafficking is regulated by cellular energy status via ampk , and this process may play a key role in inhibiting insulin secretion under low energy status . although the underlying molecular mechanisms involved in ampk - dependent katp channel trafficking remain to be elucidated , this study provides new insights into the regulation of katp channels in pancreatic -cells .
 objectiveamp - activated protein kinase ( ampk ) and the atp - sensitive k+ ( katp ) channel are metabolic sensors that become activated during metabolic stress . 
 ampk is an important regulator of metabolism , whereas the katp channel is a regulator of cellular excitability . 
 cross talk between these systems is poorly understood.research design and methodsrat pancreatic -cells or ins-1 cells were pretreated for 2 h at various concentrations of glucose . 
 maximum katp conductance ( gmax ) was monitored by whole - cell measurements after intracellular atp washout using atp - free internal solutions . 
 katp channel activity ( npo ) was monitored by inside - out patch recordings in the presence of diazoxide . 
 distributions of katp channel proteins ( kir6.2 and sur1 ) were examined using immunofluorescence imaging and surface biotinylation studies . 
 insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.resultsgmax and npo in cells pretreated with glucose - free or 3 mmol / l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol 
 / l glucose solutions . 
 immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of kir6.2 without affecting the total cellular amount . 
 increases in gmax and the surface level of kir6.2 were inhibited by compound c , an ampk inhibitor , and siampk transfection . 
 the effects of glucose deprivation on katp channels were mimicked by an ampk activator . 
 glucose deprivation reduced insulin secretion , but this response was attenuated by compound c.conclusionskatp channel trafficking is regulated by energy status via ampk , and this mechanism may play a key role in inhibiting insulin secretion under low energy status . 
RESEARCH DESIGN AND METHODS
INS-1 cells and rat pancreatic -cell culture.
Electrophysiology.
Fluorescence staining of K
Surface biotinylation and Western blotting.
Measurement of insulin secretion from pancreatic islets.
Statistical analysis.
RESULTS
Pretreatment in low glucose solution increases K
AMPK activation underlies GD-induced increases in K
AMPK increases surface levels of K
Effects of compound C and AICAR on insulin secretion during glucose deprivation.
DISCUSSION
Supplementary Material