glucokinase ( gk ) ( ec 2.7.1.1 ) is expressed in glucosensitive cells of the pancreas , liver , hypothalamus , anterior pituitary gland , and enteroendocrine k and l cells [ 13 ] .
gk has a prominent role as a glucose sensor because of its specific kinetic properties , including an affinity for glucose that is within the physiological plasma concentration range ( half - saturation level for glucose [ s0.5 ] of ~8  mmol / l ) , positive cooperativity , and lack of inhibition by glucose-6-phosphate . in liver and brain , gk is controlled by an endogenous inhibitor , the glucokinase regulatory protein ( gkrp ) [ 4 , 5 ] . during starvation , the enzyme
after refeeding , glucose mediates the dissociation of the gk - gkrp complex and gk translocates to the cytoplasm .
also , several naturally occurring activating mutations have been described that induce a conformational change in gk , which results in higher affinity for glucose , and reduce the interaction between gk and gkrp [ 6 , 7 ] .
similarly , synthetic gk activators ( gkas ) enhance gk activity and induce its translocation to the cytosol .
there is evidence that a defect in gkrp regulation of gk , resulting in increased liver gk activity probably as a result of increased cytosolic translocation , has consequences on glucose and triglyceride homeostasis in humans , even though these patients have reduced risk to type 2 diabetes [ 13 , 14 ] .
we aimed to study the metabolic consequences of gk - gkrp deregulation by overexpressing a gk activating mutant ( gka456v ) in the liver of insulin - deficient mice ( lacking endogenous gk ) .
transfected gk456v is maintained in the cytosol of hepatocytes    in vivo leading to improved glycemia in the absence of dyslipidemia .
these data provide novel insights into the consequences of gk - gkrp derangement and widen the scope for gka research in the liver .
site - directed mutagenesis to generate gk - a456v cdna was performed using the quickchange site - directed mutagenesis kit ( strategene ) , and specific primers containing the desired mutation ( 3ctggtctctgcggcggcctgcaagaag5 and 5gaccagagacgccgccggtgcaagaacg3 ) from dharmacon .
the cdnas for gk and gk - a456v were cloned into ecori - bglii of a pcaggs vector which contains the cag ( cytomegalovirus immediate - early enhancer - chicken -actin hybrid ) promoter .
the resulting plasmids pgk and pgka456v were amplified in e. coli jm109 cells and extracted using nucleobond pc 2000  ef mega prep kit ( macherey - nagel ) .
dna for animal injection was digested with sali and hindiii which cut on both sides of the expression cassette [ 16 , 17 ] in order to extend expression in vivo .
expression plasmids were transfected in huh7 cells for kinetic analysis ( supplementary figure 1 available at doi : 10.1155/2011/707928 ) .
male icr mice purchased from harlan interfarma iberica s.l were maintained in a constant 12-hour light - dark cycle and fed standard rodent chow and water ad libitum .
all animal protocols were approved by the ethics committee at the university of barcelona . to obtain diabetic animals ,
mice weighing approximately 22  g were injected i.p . with a single dose of streptozotocin ( stz ) ( 200  mg / kg ) dissolved in 100  mm citric / citrate buffer , ph 4.5 .
digested plasmid dna was introduced into murine livers using a hydrodynamic - based gene transfer technique via rapid injection of a large volume of dna solution through the tail vein .
briefly , 60  g / mouse dna was diluted in 2.0 to 2.5  ml of apyrogenic ringer - lactate solution ( fresenius kabi ) ( 0.1  ml / g body weight ) and injected into the tail vein using a 27-gauge needle and syringe within 5 to 10 seconds .
typically , the mice recovered from the injection within 2 to 10  min . at the end of the experiment , animals were sacrificed under xylazine / ketamine anesthesia .
blood samples were taken by inferior cava vein puncture and serum was obtained by centrifugation at 700  g at 4c for 15  min .
liver was dissected , snap frozen in liquid nitrogen , and subsequently stored at 80c until analysis . for gk immunodetection ,
livers from 5-hour fasted animals were fixed in a 4% pfa solution and frozen in oct compound .
7  m cryosections were obtained in a cryostat and immunostained with a 1  :  50 dilution of gk antibody ( ap7901c , abgen ) followed by an anti - rabbit antibody alexa fluor 488 ( molecular probes ) .
blood glucose levels were measured using a glucocard memory 2 apparatus ( menarini ) from a blood drop collected from the tail tip .
the veterinarian clinical biochemistry service from the veterinary hospital of the universitat autnoma de barcelona , bellaterra , spain , measured serum metabolites .
cdna synthesis was performed using ready - to - go you - prime first strand beads ( amersham biosciences ) with random hexamers .
mrna levels of the transgenic rat gk ( r - gk ) were quantified with specie - specific primers ( applied biosystems ) and normalized using -2-microglobulin as an internal control in an ht7700 real time - pcr system ( applied biosystems ) .
mrna levels of selected genes of interest were measured running a low density array ( applied biosystems ) in an ht7900 real time - pcr system ( applied biosystems ) .
frozen liver samples were homogenized in ice - cold homogenization buffer ( hepes 50  mm ph 7.4 , kcl 100  mm , edta 1  mm , mgcl2 5  mm , dtt 2.5  mm ) using a polytron pt 3000 and centrifuged at 100,000  g for 1 hour at 4c .
supernatants were used for the determination of gk activity following the spectrophotometric method described by davidson and arion .
briefly , the assays were carried out in a solution containing hepes 50  mm ph 7.4 , kcl 100  mm , mgcl2 7.5  mm , nad 1  mm , atp 5  mm , bsa 1% and indicated amounts of glucose .
the reactions were initiated by adding 5  u / ml of glucose-6-phosphate dehydrogenase ( g6pdh ) from leuconostoc mesenteroides ( roche ) and the rate of increase in absorbance at 340  nm was measured .
the gk activity was calculated as the difference between the kinase activities measured at different glucose concentrations and 0.5  mm glucose ( hexokinase activity ) , and was expressed as munits / mg liver .
frozen liver samples were homogenized in ice - cold buffer , which contained hepes 50  mm ph 7.4 , kcl 100  mm , edta 1  mm , mgcl2 5  mm , dtt 2.5  mm , and 1% pluronic f-127 ( calbiochem ) , and centrifuged at 20,000  g for 20  min at 4c .
g6pdh activity was assayed by measuring the rate of nadph production from nadp 0.5  mm and glucose-6-phosphate 2  mm , at ph 7.6 .
frozen tissue was homogenized in radioimmunoprecipitation assay buffer ( ripa ) supplemented with protease and phosphatase inhibitors and centrifuged at 15,000  g for 15  min at 4c .
30  g of tissue extract was resolved by sds - page ( 812% gel ) and electrotransferred onto a hybond - p membrane ( amersham biosciences ) .
/ fructose-2,6-biphosphatase ( ub - pfk2 ) and l - pyruvate kinase ( l - pk ) ( gifts from ramon bartrons , barcelona university ) , and carbohydrate response element binding protein ( chrebp ) ( novus biologicals ) . acc and acc - p ser-79 ( upstate ) were used at 1/2000 .
sheep anti - pepck - c antiserum ( a gift from daryl granner , vandervilt university , usa ) was used at 1  :  20,000 dilution .
all membranes were normalized using monoclonal anti--tubulin ( sigma ) at 1  :  10,000 dilution .
horseradish peroxidase activity linked to secondary antibody was detected with ecl substrate ( pierce ) in a fujifilm las 300 intelligent dark box iv imaging system .
densitometry was performed using multi gauge software . to measure hepatic free glucose , glucose-6-phosphate ( g6p ) , and lactate
briefly , perchloric acid 10% was added to frozen pieces of liver ( 5  :  1 v  :  w ) and the mixture was homogenized in a potter elvehjem apparatus at 1,500  rpm .
the homogenate was then centrifuged at 20,000  g for 10  min at 4c .
the supernatant was brought to ph 7 with k2co3 4  n and centrifuged again to remove the kclo4 formed during neutralization .
g6p was measured following the spectrofluorometric method described by lang & michal , which is based in the reaction catalyzed by g6pdh .
briefly , the assay solution contained triethanolamine 0.2  m ph 7.6 , nadp 0.2  m , mgcl2 5  mm , and g6pdh 0.17  u / ml .
lactate was determined by a spectrophotometric method based on the reaction catalyzed by lactate dehydrogenase , following gutmann & wahlefeld . briefly , the reaction cuvette contained hydrazine / glycine buffer 0.25  m/0.7  m , ph 9.3 , edta 0.15  mm , nad 2  mm , and 35  u / ml of lactate dehydrogenase . for fructose-2,6-bisphosphatase ( f2,6bp ) determination , frozen livers were homogenized in 0.1  m naoh , heated to 80c for 15  min , and centrifuged at 12,000  g for 5  min .
f2,6bp was determined in supernatants by its ability to activate pyrophosphate - dependent 6-phosphofructo-1-kinase from potato tubers , as described by van schaftingen et al . .
the hepatic triacylglyceride ( tag ) content was measured in 3  m koh , 65% ethanol extracts using a tag kit ( sigma ) , based on the method of salmon and flatt for liver saponification .
a solution of u - c - glucose and glucose was intraperitoneally injected into 5-hour fasted stz - diabetic mice ( 0.2  mg glucose , 10  ci per animal ) .
blood , liver , brain , and muscle were obtained 30 minutes after injection and dissolved into solvable ( perkin elmer ) .
blood samples were bleached with edta 0.1  m and h2o2 ( 30% ) , and liver and brain samples were bleached with h2o2 ( 30% ) alone .
radioactivity was quantified by adding scintillation liquid ( ecoscint h ) and counted in a wallac 1409 - 001 counter .
pgka456v , wild - type gk ( pgk ) and an empty vector ( pcontrol ) , were introduced into the liver of streptozotocin ( stz- ) treated mice .
hydrodynamic - based delivery of plasmid dna led to specific transfection of hepatocytes around the central vein [ 23 , 24 ] , reproducing the physiologic distribution of gk in the liver acinus .
this technique induces a transient release of liver enzymes that returns to control values by the second day after injection .
consistently , transaminase levels were low at the end - point of analysis , and similar in all treatment groups ( pcontrol 207.5  29.88  u / l , pgk 121.0  48.43 
u / l , pgka456v 117.5  40.35  u / l ; n = 49 ) . at the time of injection mice were hyperglycemic ( > 350  mg / dl ) and their insulin level was undetectable ( < 0.1 
forty - eight hours postinjection the expression of transfected rat gk and gka456v was detected by rt - pcr ( data not shown ) .
further confirmation of gk overexpression was obtained by western blot from liver extracts ( figure 1(a ) ) and gk activity assay ( figure 1(b ) ) .
gk activity was undetectable in pcontrol transfected livers irrespective of glucose concentration , as expected in long - term stz diabetic animals .
in contrast , both pgk and pgka456v transfected groups had similar gk activity at 100  mm glucose ( mu / g fresh ) , demonstrating that both treatment groups had similar amounts of gk protein .
consistent with the s0.5 for glucose of gka456v , liver extracts from pgka456v - injected animals had twice the gk activity of livers from pgk - injected animals at physiological glucose concentrations ( 5  mm ) ( figure 1(b ) ) .
gkrp retains gk in the nucleus at low glucose concentrations and releases gk when insulin , high glucose , or fructose-1p are present [ 26 , 27 ] .
different studies have suggested that the a456v mutation impairs gkrp regulation in vitro [ 6 , 28 ] .
therefore , we assessed gkrp - dependent nuclear sequestration in primary hepatocytes from stz - treated rats transfected with pgk and pgka456v , in the presence or absence of pgkrp .
densitometry analysis of western blots from nuclear and cytosolic fractions showed that cellular gka456v distribution was not affected by the presence of gkrp ( data not shown ) .
pgk - treated mice showed gk immunoreactivity throughout the liver , especially in nuclei ( figure 2 ) .
these data demonstrates , that impaired gkrp binding to gka456v changes its compartmentalization in vivo .
the impact of both gk enzymes on glucose metabolism was first evaluated in fed animals .
both gk and gka456v slightly reduced glycemia ( figure 3(a ) ) . to explore the fate of glucose in the liver
fed pgk - treated mice showed significantly higher f2,6bp and glycogen contents ( figure 4(a ) ) , together with a rise in lactate , indicating a higher glycolytic flux .
gka456v - expressing livers also showed higher hepatic free glucose than both control and gk groups , even though the content of g6p was similar in all three groups .
glycogen content was significantly higher than control but similar to pgk - treated group ( figure 4(a ) ) .
pgka456v - treated animals showed a marked reduction of glycemia after a 5-hour fast ( figure 3(a ) ) .
lactate was only increased after pgk treatment ( pcontrol , 7.296  0.772  mm ; pgka456v , 8.778  0.649  mm ; pgk , 10.62  0.608  mm 
hepatic free glucose content was low in the pgka456v treatment group ( table 1 ) .
g6p levels were reduced to the same extent in control and gka456v groups , whereas gk - expressing animals showed similar g6p and atp concentrations to well - fed animals .
glycogen content in gk - expressing animals was significantly higher than control and gka456v - expressing groups .
fed - to - fast transition produced a marked reduction of glycogen stores in pgka456v - treated animals as compared to pcontrol and pgk , suggesting an increased rate of glycogenolysis in gka456v - expressing animals ( figure 4(b ) ) . in order to clarify whether glucose distribution in vivo favored the liver , we quantitated c radioactivity derived from uptake and metabolism of uc - glucose in various tissues 30 minutes after injection .
c label was increased exclusively in the liver of both pgk and pgka456v treatment groups ( control , 100  7.88% ; gk , 138.73  14.63% ; gka456v 144.1
 8.99% ; p = 0.037 gk versus control and p = 0.003 pgka456v versus control ) . to assess whether pgka456v expression would affect glucose clearance in healthy mice
, we performed a glucose tolerance test 1 week after transfection ( figure 3(b ) ) .
consistently , our data demonstrates a significant increase in serum tag ( pcontrol 33.5  7.72  mg / dl , pgka456v 38.13  7.66  mg / dl , pgk 74.00  14.76  mg / dl , p < 0.05 versus pcontrol and pgka456v ) and nefa ( pcontrol 0.325  0.09  mm , pgka456v 0.4013  0.05  mm , pgk 0.66  0.10  mm p < 0.05 versus pcontrol ) only in the postabsorptive state .
besides , gk overexpressing mice showed a tendency to increase hepatic tag content , which is consistent with the literature [ 30 , 31 ] .
in contrast , gka456v overexpression did not promote dislipemia , as evidenced by maintained levels of circulating nefa and tag .
there was a significant increase in -hydroxybutyrate levels in the pgka456v group ( pcontrol , 0.18  0.03  mm ; pgka456v , 0.27  0.03  mm 
in order to elucidate the mechanism responsible for the metabolic profile observed , we analyzed protein and mrna levels for key enzymes and transcription factors . both gk and gka456v treatments
were accompanied by increases in l - pk protein ( figures 5(a ) and 5(b ) ) .
pgk - treatment resulted in higher levels of ubiquitous pfk2 ( pfkfb3 ) protein ( figures 5(a ) and 5(b ) ) , but not mrna ( table 2 ) , consistent with elevated f2,6bp observed in this treatment group ( table 1 ) . also , endogenous
, mouse gk mrna expression was induced in the livers of pgk - injected mice , as detected by species - specific qrt - pcr .
correlating with the induction of the expression of endogenous gk , cmyc mrna was only increased in pgk - treated animals .
other regulatory factors implicated in the regulation of endogenous gk expression , such as srebp and lxr , were not affected by treatment with pgk and pgka456v ( table 2 ) . as an increment in gk expression in the liver
is commonly associated with increased lipogenesis [ 32 , 33 ] , enzymes and transcription factors involved in de novo lipogenesis were analyzed .
gk- and pgka456v - expressing groups presented increased mrna content for fasn and mod1 that were more pronounced in pgk - treated animals .
also , acc1 and chrebp were more induced by the overexpression of gk than by gka456v .
gk , through the production of xylulose-5p , mediates the inhibition of gluconeogenesis by glucose [ 34 , 35 ] .
f2,6bp   has also been associated with the inhibitory effect of glucose metabolism on gluconeogenesis .
consistently , we observed a reduction of pepck at the protein level in both pgk- and pgka456v - treated mice .
similarly , pgc-1 and hnf4 mrnas were reduced in pgk - injected mice , whereas gka456v - overexpressing livers only showed reduced pgc-1 mrna . in contrast , the level of mrna for the catalytic subunit of glucose-6-phosphatase ( glc6pase ) were strikingly higher in gka456v - overexpressing animals , consistent with more glycogen breakdown observed after a 5-hour fast .
similar results were observed in healthy mice overexpressing pgka456v after a short fast ( data not shown ) .
to evaluate the consequences of gk activation and deregulation by gkrp in the liver , we hydrodynamically injected pgka456v in the liver of stz - diabetic mice .
this strategy allowed us to obtain a mouse model where the effects of exogenous gks are isolated from those of endogenous gk ( undetectable levels of mouse gk mrna and activity in pcontrol - treated animals ) and insulin action ( insulin levels
in addition , the use of hydrodynamic gene transfer provides proper zonal expression of gk and gka456v in perivenous hepatocytes [ 23 , 25 ] .
therefore , only animals that were positive for exogenous expression of gk as determined by qrt - pcr ( 3x sd of controls ) were selected for further analysis ( data not shown ) .
after selection , both gk - overexpressing groups had identical protein levels and maximal activity ( 100  mm glucose ) figures 1(a)1(b ) . these results are not in agreement with the reported instability of liver gk protein in a phhi - gka456v transgenic mouse   and the gkrp knock - out model [ 37 , 38 ] , probably because of the lack of endogenous transcriptional control in pgka456v . also , gka456v activity at 5  mm glucose was higher in liver extracts , as expected from the kinetic changes associated with the mutation .
the gka456v mutation resides on a hinge of the enzyme responsible for the conformational changes associated with gkrp interaction and allosteric activation .
gkrp regulates gk action in hepatocytes by retaining the enzyme in the nucleus at low glucose concentrations and liberating gk when insulin , high glucose levels , or fructose-1p is present [ 26 , 27 ] .
we show here that gka456v is predominantly cytosolic in the liver of transfected animals , contrary to the expected nuclear compartmentalization of wild - type gk .
in addition , gka456v was not appropriately translocated to the nucleus of diabetic primary hepatocytes in the presence of excess gkrp ( data not shown ) .
lack of gkrp binding to gka456v has been shown by heredia et al .   in vitro and inferred from the structural characterization of gk bound to synthetic activators [ 12 , 39 ] .
recent reports on the p446l mutation in gkrp also suggest that a decrease in gk inhibition results in increased gk activity , suggesting that gk localizes to the cytosol .
an analysis of gk and gka456v overexpression in well - fed animals demonstrated small but significant changes in glycemia , together with increased hepatic glucose metabolism , as suggested by higher f2,6bp and glycogen content , in the absence of alterations to lipid serum metabolites .
no changes were observed in enzymes or regulatory proteins involved in gluconeogenesis in fed animals ( data not shown ) .
[ 31 , 33 , 40 ] , who used a low dose of gk - expressing adenovirus ( expressed largely in periportal hepatocytes , [ 41 , 42 ] ) but differ from those obtained using high doses of adenovirus , indicating that dose and/or zonal expression of gk influences the metabolic impact of gk overexpression . when animals were examined after a short fast ( postabsorptive ) , marked differences were observed among groups .
( 1 ) gk overexpression reduced glycemia ~15% , consistent with enhanced glucose metabolism in the liver , as shown by increased g6p , f2,6bp , glycogen content , and glycolytic and lipogenic enzymes together with higher serum lactate , nefa and tag concentrations .
have shown that a fed - to - fast transition in stz - diabetic mice results in increased gluconeogenic potential and fatty acid oxidation in the liver , consistent with results obtained in pcontrol - treated animals in the present study .
conversely , pgk treatment resulted in reduced pepck and pgc1 mrna , supporting the view that gk overexpression indirectly inhibits gluconeogenesis and fatty - acid oxidation . consistently , we measured higher nefa and tag serum levels in the postabsorptive state although no changes in -hydroxybutyrate were observed .
these results indicate that enhanced glucose metabolism resulting from gk overexpression had a small effect on glycemia , possibly because pgk dose was low .
( 2 ) gka456v overexpression led to a significant reduction of serum glucose of ~60% that was consistent with higher glucose clearance on a glucose tolerance test in fasting , healthy mice .
fasting hypoglycemia is also observed in human carriers of the p446l gkrp mutation [ 14 , 44 ] .
gka456v overexpression did not impact the concentration of serum lipids whereas -hydroxybutyrate was increased , contrary to the dyslipidemia observed in gk overexpressing animals in this and earlier studies , including human carriers of the p446l gkrp mutation [ 14 , 44 ] .
hepatic levels of f2,6bp and l - pk protein were increased but other indicators ( g6p , fasn mrna , chrebp protein , and mrna and glycogen content ) of glucose utilization were lower than in gk - expressing livers .
importantly , glycogen balance ( fed - to - fast ) was significantly lower in gka456v - expressing animals , indicating an important net loss of glycogen .
g6pase expression was also higher in this group of animals , providing an explanation for increased glycogen loss observed in gka456v - containing livers .
the involvement of g6pase in the positive regulation of glycogenolysis has been shown in several animal models overexpressing p36 or p46 components of the g6pase system , resulting in decreased glycogen levels in the hepatocyte [ 4548 ] . moreover
, a lack of g6pase system activity observed in glycogen storage disease type 1a , or in a g6pase ko promotes glycogen accumulation .
activation of the nonoxidative branch of the pentose phosphate pathway , as a result of high exogenous glucose , transiently upmodulates g6pase mrna levels and stability in an insulin - independent manner [ 34 , 35 ] .
gka456v - expressing livers showed higher g6pdh activity and gsh / gssg ratio during the fed - to - fast transition than control or gk groups ( figures 4(c)-4(d ) and table 1 ) .
increased g6pase mrna and protein in the presence of gka456v would suggest an induction of glucose cycling .
it is , however , clear that this counter regulation shall have consequential effects on glucose homeostasis if persistent , as occurs in the gka456v model .
all in all , the present data confirms that pgk overexpression in the liver results in relatively small changes in glycemia but overt dislypidemia .
the present model represents the first attempt to overexpress pgk in perivenous hepatocytes , and confirms previous observations using adenovirus .
in contrast to the work of scott et al .   and morral et al .
, gk overexpression in perivenous hepatocytes does not significantly affect glc6pase expression , suggesting that zonation is an important experimental variable not sufficiently addressed to date in the field .
in addition , we have demonstrated that gka456v overexpression reduces blood glucose to a great extent , probably due to its capacity to induce glc6pase .
we conclude that impaired gkrp regulation and gka456v altered kinetics greatly influence liver metabolism , in line with results obtained in humans with a mutant gkrp .
besides , it suggests that activating gk exclusively in the liver could be a feasible strategy for diabetes , widening the scope for gka research .
similar conclusions , based on its potent induction of glucose clearance by the liver , have been obtained after liver - specific gk activation in chicken in a recent study by rideau et al . .
studies on animal models of type 2 diabetes are under way in our laboratory in order to evaluate the metabolic impact in the context of insulin resistance and obesity .
 recent reports point out the importance of the complex gk - gkrp in controlling glucose and lipid homeostasis . 
 several gk mutations affect gkrp binding , resulting in permanent activation of the enzyme . 
 we hypothesize that hepatic overexpression of a mutated form of gk , gka456v , described in a patient with persistent hyperinsulinemic hypoglycemia of infancy ( phhi ) and could provide a model to study the consequences of gk - gkrp deregulation in vivo . 
 
 gka456v was overexpressed in the liver of streptozotocin diabetic mice . 
 metabolite profiling in serum and liver extracts , together with changes in key components of glucose and lipid homeostasis , were analyzed and compared to gk wild - type transfected livers . 
 cell compartmentalization of the mutant but not the wild - type gk was clearly affected in vivo , demonstrating impaired gkrp regulation . 
 
 gka456v overexpression markedly reduced blood glucose in the absence of dyslipidemia , in contrast to wild - type gk - overexpressing mice . 
 evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver . 
 pepck mrna was not affected , whereas the mrna for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of gka456v - treated animals , suggesting that glucose cycling was stimulated . 
 our results provide new insights into the complex gk regulatory network and validate liver - specific gk activation as a strategy for diabetes therapy . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion