human ether - a - go - go - related gene ( herg ) encodes the herg ion channel , which is a member of the voltage - dependent potassium channel ( kv ) family .
herg mutations reduce the outward flow of potassium during repolarization and elongate the qt interval leading to polymorphic ventricular tachycardia , cardiac syncope , and sudden death .
herg ion channels are expressed in the human pancreas where it has been shown to negatively regulate insulin secretion and positively regulate glucagon secretion .
fluoroquinolones are antibiotics that are effective against gram - negative bacilli , but as drug development continues , their antibacterial spectra expand .
fourth - generation fluoroquinolones , such as moxifloxacin and levofloxacin , are also effective against streptococcus pneumoniae , a gram - positive cocci , and anaerobic bacteria . with the widespread clinical use of these drugs , dysglycemia has been reported as an adverse effect .
gatifloxacin , ofloxacin , and lomefloxacin   have been reported to lead to hypoglycemia , whereas ofloxacin   and gatifloxacin   have been reported to result in hyperglycemia .
although these side effects are rare , they require hospitalization and can endanger the life of a patient ; thus , patients taking fluoroquinolones should be monitored for dysglycemia . some fluoroquinolones cause hypoglycemia by blocking atp - sensitive potassium channels ( katp ) in pancreatic -cells leading to stimulation of insulin secretion . by stimulating histamine secretion
gatifloxacin can also cause vacuolization of islet -cells leading to a decrease in insulin production and hyperglycemia .
katp channels and other ion channels have been found to synergistically regulate insulin secretion ; thus , herg potassium channels and katp channels together may regulate insulin secretion by pancreatic -cells . because the ability of moxifloxacin to inhibit herg channels is unknown ,
this study explored the effect of moxifloxacin on herg channel currents and glucose metabolism in mice .
c57bl/6 herg knockout mice were created using transcription activator - like effector nucleases ( talens ) .
this study was approved by the ethics committee of beijing tongren hospital , capital medical university , and was performed according to the principles of the declaration of helsinki ii .
animal experiments followed the national ethical guidelines implemented by our institutional animal care and use committee and were approved by the ethical review committee at the institute of zoology , capital medical university , china .
human pancreatic tissues were fixed by immersion in 4% paraformaldehyde for 2  h at 4c and were then embedded in paraffin .
paraffin sections ( 5  m ) were pretreated for antigen retrieval using a microwave .
paraffin sections were incubated with primary antibody , goat anti - insulin antibody ( diluted 1  :  100 , santa cruz biotechnology inc . , ca , usa ) or rabbit anti - herg antibody ( diluted 1  :  200 , santa cruz biotechnology inc . ,
the sections were washed three times with phosphate - buffered saline ( pbs ) and then were incubated with alexa fluor 555 donkey anti - goat - cy3 ( diluted 1  :  200 , santa cruz biotechnology inc . , ca , usa ) or alexa fluor 488 goat anti - rabbit - cy2 ( diluted 1  :  200 , santa cruz biotechnology inc . ,
images were taken with a laser scanning confocal microscope ( leica tcssp5 ; leica , wetzlar , germany ) .
human embryonic kidney ( hek ) 293 cells were provided by the cell research center at basic medical peking union medical college .
hek293 cells were cultured in a 37c incubator with 5% co2 and humidified air . the target gene , herg cdna , and the expression vector , gv314 ( genechem co. , shanghai , china ) , were transfected into cells using lipofectamine 2000 ( invitrogen , carlsbad , ca , usa ) according to the manufacturer 's protocol .
vector gv314 was cotransfected because it contains the marker gene gfp , sv40 , cmv promoter , and ampicillin resistance gene .
cells were cultured in dulbecco 's modified eagle 's medium ( dmem ; gibco , md , usa ) , 10% fetal bovine serum ( gibco , md , usa ) , 1% penicillin - streptomycin ( gibco , md , usa ) , and 1% sodium pyruvate ( gibco , md , usa ) for 4872  h after transfection .
cells exhibiting strong fluorescence microscopically ( te2000-u , niko , japan ) were selected for patch clamp experiments . a patch clamp amplifier ( epc-10 , heka , germany )
hek293 cells were bathed in 137  mmol / l of nacl , 4  mmol / l of kcl , 2  mmol / l of cacl2 , 1  mmol / l of mgcl2 , 10  mmol / l of hepes , and 10  mmol / l of glucose , ph 7.4 .
when the patch pipette was filled with the internal pipette solution , the pipette had a resistance of 2.54.0  m. the internal pipette solution contained 130  mmol / l of kcl , 5  mmol / l of mgatp , 1  mmol / l of mgcl2 , 5  mmol / l of egta , and 10  mmol / l of hepes , ph 7.3 .
after membranes were ruptured using negative pressure suction , resistance ranged from 500 to 600  m. experiments were performed from 25 to 28c .
louis , mo , usa ) was dissolved to a concentration of 10  mmol / l with double - distilled water .
this study contained four groups : wild - type mice that received physiological saline , wild - type mice that received moxifloxacin , herg knockout mice that received saline , and herg knockout mice that received moxifloxacin .
the control groups received physiological saline at the same volume via the same route as the test groups .
mice were fasted overnight for 16  h but were allowed free access to water .
they were given 200  mg / kg of moxifloxacin or the same volume of physiological saline via gavage , and then they were given 2  mg / kg of glucose by intraperitoneal injection .
blood samples were taken from the angular vein at 0 , 15 , 30 , 60 , and 120  min after glucose injection .
blood glucose concentrations were assessed using an automatic glucometer ( one touch , lifescan , usa ) .
insulin concentrations were measured using a highly sensitive mouse insulin immunoassay kit ( antibody and immunoassay services , hku , china ) according to the manufacturer 's protocol .
we used a dose - response curve fitted to a hill equation , y = [ ( a1  a2)/(1 + ( x / c)nh ) ] + a2 , in which a1 represents 0 tail current inhibition , a2 represents 100% inhibition , c represents the ic50 concentration , and nh represents the hill slope .
an independent sample t - test was used to compare two sets of data . a one - way anova followed by tukey 's or dunnett 's test
pancreatic -cells , which are located in pancreatic islets , were visualized by insulin immunoreactivity ( figure 1(b ) ) .
herg protein - positive cells were located in these islets ( figure 1(c ) ) .
colocalization of insulin and herg by immunostaining indicated that herg is expressed in pancreatic -cells ( figure 1(d ) ) .
hek293 cells were held at 80  mv for 1  s and then were depolarized for 3  s using 10  mv intervals from 60  mv to + 50  mv .
the current initially increased and then decreased , in a time - dependent manner , as the voltage increased .
a tail current was elicited for 3  s at 40  mv and the peak tail current at each voltage represented the degree to which the herg channel was open at that voltage .
after perfusion of the transfected hek293 cells with 100  mol / l of moxifloxacin for 5  min , the current was reduced ( figure 2(a ) ) .
curves i - v indicated that 100  mol / l of moxifloxacin significantly reduced the time - dependent and peak tail currents when the voltage was greater than 0 and 10  mv , respectively ( figure 2(b ) ) .
cells were depolarized to + 20  mv for 3 s and peak tail currents were recorded at different moxifloxacin concentrations ( 10 , 100 , and 1000  mol / l ) .
tail currents gradually decreased with the increasing concentrations of moxifloxacin indicating that inhibition by moxifloxacin was concentration - dependent .
herg channel currents were completely inhibited at 1000  mol / l of moxifloxacin ( figure 2(c ) ) . by plotting moxifloxacin concentration ( 100  mmol / l ) on the  x - axis and tail current inhibition ( % ) on the  y - axis ,
a dose - response curve was generated and the data were fitted to a hill equation , y = [ ( a1  a2)/(1 + ( x / c)nh ) ] + a2 .
mol / l and the hill slope , nh , was 0.96 ( figure 2(d ) ) . blood glucose and insulin concentrations of mice that underwent the glucose tolerance test
200  mg / kg of moxifloxacin led to a reduction in the blood glucose concentration at 60  min ( p = 0.0346 ) ( figure 3(a ) ) .
insulin levels in the moxifloxacin group were higher than those in the saline group at 60  min ( p = 0.0373 ) ( figure 3(b ) ) .
the areas under the blood glucose concentration versus time curve and the insulin concentration versus time curve differed ( p = 0.0383  and 0.0383 , resp . )
there were no significant differences in blood glucose or insulin concentrations in herg knockout mice treated with moxifloxacin or saline ( figures 3(c ) and 3(d ) ) .
increased glucose levels trigger a series of reactions that ultimately lead to insulin secretion by islet -cells . the transformation and utilization of glucose increase the intracellular ratio of atp / adp causing katp channels to close
the cell membrane is depolarized causing voltage - dependent calcium channels to open , which leads to an increase in the concentration of free intracellular calcium .
in addition to katp channels , other voltage - gated potassium channels are expressed in pancreatic -cells and regulate insulin secretion by repolarizing membranes .
were the first to report that herg potassium channels are expressed in human islets -cells , and they studied the effect of herg channels on insulin secretion using electrophysiological and molecular techniques .
.   also found herg in human islets using reverse transcription - polymerase chain reaction ( rt - pcr ) , but they did not determine its specific distribution .
the herg inhibitors e-4031 , dofetilide , and verapamil impact channel gating to various degrees and can cause currents to be reduced or completely inhibited .
basal insulin secretion in ins-1 -cells was shown to increase upon blockage of erg channels by e-4031 .
we demonstrated that the time - dependent and tail currents for herg decreased in response to 100  mol / l of moxifloxacin indicating that moxifloxacin inhibits the herg channel in hek293 cells .
as moxifloxacin concentrations increased , tail currents were further reduced and completely inhibited at 1000  mol / l of moxifloxacin .
we found that moxifloxacin inhibition was concentration - dependent and that the ic50 was 36.65 
fluoroquinolones can induce life - threatening dysglycemia , but , in the clinical concentration range , fluoroquinolones do not initiate dysglycemia ; they rather enhance insulin secretion , which is induced by high glucose concentrations .
fluoroquinolones can block herg channels and pancreatic -cell katp channels ; however , the structures of the fluoroquinolones that inhibit herg channels and katp channels differ .
the amino group at position c5 of fluoroquinolones plays a major role in blocking herg channels , whereas the substituents at positions n1 , c7 , and c8 of fluoroquinolones are responsible for the inhibition of katp channels .
inhibition of katp channels is required for fluoroquinolone - induced hypoglycemia , but channel inhibition alone does not sufficiently explain this side effect .
when herg channels were selectively blocked by perfusion of islets with antiarrhythmic drugs , insulin secretion increased in the presence of glucose and arginine .
a k current that is pharmacologically and biophysically similar to the herg current has been reported .
these data suggest that herg channels contribute to the regulation of insulin secretion by human pancreatic -cells .
patients receiving insulin concomitant with moxifloxacin have a significantly higher risk of hypoglycemia in taiwan .
the fluoroquinolone moxifloxacin has been associated with the highest risk of hypoglycemia followed by levofloxacin and ciprofloxacin .
.   found that hypoglycemia occurred most often with moxifloxacin compared to levofloxacin and ciprofloxacin .
ishiwata et al .   reported that intravenous injection of 100  mg / kg of moxifloxacin increased the serum glucose concentration and temporarily elevated serum histamine and epinephrine concentrations in fasted rats .
serum glucose , epinephrine , and histamine concentrations remained unchanged when moxifloxacin was administered at 75  mg / kg . serum insulin concentrations were not altered by moxifloxacin at both 75 and 100  mg / kg .
this suggests that moxifloxacin can cause hyperglycemia by inducing histamine and epinephrine release . in our study , 200  mg / kg of moxifloxacin reduced blood glucose levels in wild - type mice 60  min after the start of the glucose tolerance test ; this reduction may be a result of a slight increase in insulin secretion .
in contrast , moxifloxacin did not significantly alter blood glucose or insulin levels in herg knockout mice .
serum glucose levels increased and insulin concentrations decreased in herg knockout mice . in a word , this is suggesting that moxifloxacin reduces blood glucose levels and increases insulin secretion via the herg protein .
it is also not known whether other channels participate in the moxifloxacin - induced hypoglycemic effect .
 the purpose of this study was to investigate the effect of moxifloxacin on herg channel protein and glucose metabolism . 
 herg expression was investigated using immunohistochemistry . 
 the whole - cell patch clamp method was used to examine the effect of moxifloxacin on herg channel currents . 
 a glucose tolerance test was used to analyze the effects of moxifloxacin on blood glucose and insulin concentrations in mice . 
 results show that herg protein was expressed in human pancreatic -cells . 
 moxifloxacin inhibited herg time - dependent and tail currents in hek293 cells in a concentration - dependent manner . 
 the ic50 of moxifloxacin inhibition was 36.65  
 mol / l . 
 moxifloxacin ( 200  mg / kg ) reduced blood glucose levels and increased insulin secretion in wild - type mice at 60  min after the start of the glucose tolerance test . 
 in contrast , moxifloxacin did not significantly alter blood glucose and insulin levels in herg knockout mice . 
 serum glucose levels increased and insulin concentrations decreased in herg knockout mice when compared to wild - type mice . 
 the moxifloxacin - induced decrease in blood glucose and increase in insulin secretion occurred via the herg protein ; thus , herg protein plays a role in insulin secretion . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion