there has been a dramatic increase in the prevalence of diabetes mellitus in recent years .
the chronic effects of diabetes may manifest in macro- and microvascular complications that are the major causes of morbidity and mortality in patients with diabetes .
diabetic nephropathy ( dn ) , one of the microvascular complications , is a leading cause of death from kidney failure [ 2 , 3 ] .
apart from haemodynamic factors , hyperglycaemia has been shown to be an underlying cause of pathogenesis in dn .
the damaging effects of hyperglycaemia have been partly attributed to increased cellular glucose uptake in cells that are not protected from high ambient glucose levels .
several investigators have associated the expression of cip / kip cyclin - dependent kinase ( cdk ) inhibitors , p21 and p27 , with glomerular hypertrophy [ 57 ] .
it has been proposed that p21 and p27 may be involved in hypertrophy independently of their cell cycle regulatory properties ( monkawa 2002 ) .
furthermore , the induction of p21 and p27 is also required for senescent arrest , a molecular signature of hypertrophic changes in the early stages of the development of diabetic kidney disease .
the fact that p21 null mice do not develop glomerular hypertrophy supports the importance of p21 in dn .
the activation of the mammalian target of rapamycin ( mtor ) , a serine / threonine kinase , plays a pivotal role in the pathologic forms of hypertrophy in the kidneys [ 1012 ] .
these complexes have two different scaffolding proteins , raptor and rictor . by interacting with distinct downstream targets ,
these scaffolding proteins connect mtor to different signalling pathways , resulting in discrete functional roles .
the raptor - mtor protein complex is rapamycin sensitive ; it integrates extracellular and intracellular signals originating from growth factors , hormones , and nutrients .
this complex plays a key role in regulating the cellular response to nutrients by phosphorylating the downstream target proteins , p70s6 kinase1 ( s6k ) and initiation factor 4e .
studies on skeletal muscle cells have shown that , through a negative feedback mechanism , the activation of the mtor pathway may lead to insulin resistance .
furthermore chronic rapamycin treatment in rats induced the expression of hepatic gluconeogenic enzymes , which may adversely affect glucose levels in a diabetic state . on the other hand ,
it has been shown by several investigators that the inhibition of the mtor signalling pathway has a therapeutic potential for the treatment of dn [ 13 , 16 ] .
mtor is also regulated by amp - activated protein kinase ( ampk ) , a sensor of intracellular amp levels .
mammalian ampk is a heteromeric complex consisting of one catalytic subunit  and regulatory  ,  subunits . through a conformational change in the  subunit
, amp facilitates the phosphorylation of thr-172 on the  subunit by various upstream kinases , including ca - calmodulin - dependent kinase  , tgf--activated kinase 1 , and lkb1 serine / threonine kinase .
the antidiabetic drug metformin depletes cellular atp levels by blocking mitochondrial respiratory complex i. in turn , the elevated amp levels induce the activation of ampk .
nevertheless , it has been suggested that metformin may activate ampk through an amp - independent mechanism .
although ampk activation results in the inhibition of mtor signalling , recent findings also suggest that metformin may abolish mtor activation independently of ampk . apart from its insulin - sensitising properties
, metformin may have several beneficial effects in various clinical settings . according to clinical studies in both type 1 and type 2 diabetes ,
several studies have shown that excess glucose increases cell size in various cell types through the activation of akt - mtor signalling ; however , the mechanism leading to glucose - induced mtor activation has not been well defined .
it has been suggested that the hypertrophic changes induced by hyperglycaemia may be the consequence of mtor activation through autocrine tgf- signalling .
in addition , mtor activity has also been associated with increased expression of the glucose transporter 1 ( glut1 ) in mesangial cells .
however , saturation of glucose uptake in mesangial cells has been reported to occur at 30  mm , indicating that hyperglycaemia can induce mtor in the absence of increased glut1 expression .
the aim of this study was to compare the inhibitory effects of rapamycin and metformin on proliferation and cell growth in the context of high glucose - induced ampk / mtor signalling .
we have observed differential effects of metformin and rapamycin in several ampk / mtor - related aspects with relevance to dysregulated cell growth and cell cycling in dn .
human embryonic kidney ( hek293 ) cell line was maintained in minimum essential media ( invitrogen ) supplemented with 10% fbs , 1% pen / strep ( invitrogen ) , and 1% mem nonessential amino acid solution .
the cells were cultured at 37c in a 95% air/5% co2 environment and passaged every 3 - 4 days at subconfluence .
conditionally immortalised human podocyte cells ( courtesy of professor saleem , bristol ) were cultured in rpmi 1640 medium containing 11  mm d - glucose , 10% foetal bovine serum 1% penicillin / streptomycin .
briefly , the cell line was generated by isolating podocyte cells from a normal human kidney specimen and transfected with a temperature sensitive simian virus-40 large t - antigen .
the cells proliferate at a permissive 33c , then culturing the cells at 37c switches off t - antigen expression , allowing the cells to assume a native phenotype . in the experiments ,
the cells were cultured with different concentrations of d - glucose ( sigma - aldrich ) . where appropriate , mannitol ( sigma - aldrich ) was used to control osmotic effects .
metformin ( sigma - aldrich ) stock solution was prepared in phosphate buffered saline ( pbs ) .
eight pgipz lentiviral shrna constructs against human amp - activated , alpha2 catalytic subunit ( prkaa2 ) , and a nonsilencing construct were purchased from open biosystems .
pgipz plasmids were stored in bacterial cultures of e. coli ( prime plus ) in lb lennox ( 5  g nacl / l ) with 8% glycerol , 100  g / ml carbenicillin , and 25  g / ml zeocin . isolation of plasmid dna was done by plasmid midi kit ( qiagen ) according to the protocol supplied by the manufacturer .
stable cell lines were generated by transfecting hek293 cells with 2  g / ml plasmid dna in 24 well plates using arrest - in transfection reagent ( open biosystems ) .
selection for stably transfected cells was done in medium supplied with 8  g / ml puromycin .
the cell suspension was loaded in 96 well microtitre plates at 1  10/ml density and allowed to grow until 50% confluence when the cells were exposed to experimental conditions .
cell proliferation was determined using the cell titre96 aqueous non - radioactive cell proliferation assay ( promega ) .
the proliferation assay is an mts - based method that spectrophotometrically measures the conversion of tetrazolium salt into a formazan product .
the quantity of this product is directly proportional to the number of living cells in culture .
flow cytometry was performed to analyse cell cycle distribution and cell size in hek293 cells .
after treatment , the cells were trypsinised and washed with pbs by centrifuging them twice at 200  g for 5 minutes ( min ) .
the cells for cell cycle analysis were fixed in cold saline gm and 90% ethanol ( 1  :  3 ratio ) and stored at 80c . before analysis , the cells were centrifuged at 500  g for 5  min , resuspended in facs buffer ( pbs , 2% fcs , 10  mm sodium azide ) , and treated with 100  g / ml rnase a ( sigma ) .
dna was stained with 50  g / ml propidium iodide ( sigma ) for 1 hour ( h ) at room temperature ( rt ) , and the percentage of 1  10 cells in the g1/g0 , s , and g2/m phases cells was determined .
the cells for cell size analysis were resuspended in facs buffer and relative forward side scatter determined on a becman - coulterton epics xl.mcl flow cytometer running expo32 adc software ( 10,000 events ) . in order to investigate the viability of the cells , propidium iodide
floating and adherent cells were harvested by trypsinisation , pelleted by centrifugation at 200  g for five minutes , and then washed twice with facs buffer .
the pellet was resuspended in facs buffer with 20  g / ml propidium iodide , incubated on ice for 10 minutes , and analysed for dye inclusion on a bd accuri c6 flow cytometer .
cell proteins from at least two independent experiments were extracted by addition of lysis buffer containing 20  mm tris - hcl ( ph 7.4 ) , 150  mm nacl , 1  mm edta , 1% tx100 , and 1 tablet/10  ml phosstop phosphatase inhibitor ( roche ) .
the suspension was centrifuged at 14000  g and the supernatant containing cellular protein was collected . for western blotting ,
a 12.5% sodium dodecyl sulphate polyacrylamide gel was run under standard conditions , loading 25  g of total protein in each lane .
the gel was placed in transfer buffer and set up for transfer onto a polyvinylidene fluoride membrane at 250  ma overnight .
the membrane was rinsed in tris - buffered saline immersed in blocking buffer ( 2% bsa ) for 1  h then incubated with primary antibodies ( p - mtor ser , p - ampk thr , p - s6k thr , p21 , 1  :  1000 , and cell signalling ) overnight at 4c . after rinsing in wash buffer ,
the membrane was incubated with horseradish peroxidase - conjugated secondary antibody ( cell signalling ) for 1  h at 1  :  5000 dilution at rt .
an enhanced chemiluminescence kit ( amersham ) was used for detection of the bands . in order to control protein loading , total - mtor , and -actin antibodies
after exposure to 8  mm metformin for 24  h , the cells were fixed with 4% paraformaldehyde in pbs for 15  min at rt .
the slides were washed in pbs , permeabilized , and blocked with 0.2% triton x-100 , 10% fcs , 125  mm l - lysine , and 10% sodium azide for 30  min .
the slides were washed and incubated with the primary antibody at 1  :  400 dilution ( p21 , cell signalling ) overnight .
the cells were rinsed and subsequently incubated with alexa fluor 568 ( invitrogen ) goat anti - mouse secondary antibody at 1  :  500 and dapi at 1  :  1000 dilutions for 1  h at rt .
the slides were analysed by fluorescent microscopy ( nikon eclipse 80i microscope and a nikon digital colour camera ) .
total protein / cell number ratio was used to determine whether the alteration of cell growth in response to high glucose treatment was accompanied by cell hypertrophy .
for the experiments , hek293 cells were cultured in minimal essential medium . at the end of the treatment period
, the cells were trypsinised and washed twice with pbs and counted in a haemocytometer chamber .
the cells then were lysed to measure the total protein content by the bca protein assay ( thermo scientific ) .
the total protein / cell number ratio expressed as g/10 cells was used as a hypertrophy index .
normality of distribution was verified by shapiro - wilk test and homogeneity of variance by levene 's test .
unless otherwise indicated , statistical significance of differences was calculated by t - test or one - way anova .
in order to characterise the effects of metformin and rapamycin on hek293 cell proliferation , a dose - response study was carried out , using the mts assay . in other studies the effects of metformin and rapamycin have already been compared by using the alamar blue metabolic assay .
the investigators have found a concentration - dependent difference between the effects of metformin and rapamycin on proliferation . in order to test whether their result could be reproduced by using the mts assay , the two drugs were used in the same concentration ranges .
the results show that over a 24  h period , metformin and rapamycin inhibited cell growth in hek293 cells in a dose - dependent manner ( figure 1 ) .
however , increasing concentrations of metformin inhibited proliferation in a linear fashion , whereas increasing the concentration of rapamycin in the range of 10500  nm did not cause a significant change in cell proliferation .
although zakikhani et al .   used lower serum concentration and longer incubation time ; the findings presented here are in line with their results , showing that the inhibitory effect of metformin on cell proliferation is concentration - dependent and , at higher concentrations , more pronounced than that of rapamycin .
furthermore , 20  m compound c ( dorsomorphin ) , a research compound widely used for the inhibition of ampk had an opposing effect on metformin - induced decrease in proliferation .
the purpose of this experiment was to investigate how cell cycling is affected by metformin and rapamycin treatments , and whether cell cycle changes are modulated by high glucose concentration .
the effect of metformin on cell growth and proliferation has been investigated by various cell culture studies [ 17 , 27 , 28 ] . in these studies
the use of these relatively high concentrations of metformin may be attributed to the low uptake of this drug by immortalised cell lines . in the context of the inhibition of hepatic gluconeogenesis
, the use of metformin in the millimolar range has been shown to be physiologically relevant .
the inhibitory effect of 8  mm metformin on cell cycle progression has been demonstrated in a cell culture study of breast cancer cells . in susceptible cells
this concentration caused an approximately 50% reduction in cell number over a 24  h culture period .
as demonstrated in figure 1 , at 8  mm concentration , metformin caused an approximately 50% reduction in proliferation , suggesting that our findings may be comparable to earlier studies . in this experiment metformin
was used at 8  mm and its effect on cell cycling was compared to that of rapamycin .
the effect of rapamycin on proliferation has also been studied in vascular smooth muscle cells .
the inhibitory effect of rapamycin on proliferation has been shown in a range of concentrations ( 1100  ng / ml ) .
it has also been reported that rapamycin did not affect the viability of cultured cells in this concentration range . in order to maximise its effect on cell cycling , in this experiment rapamycin
it has also been shown by several investigators that after 24 to 72  h high glucose induces cellular changes implicated in the development of diabetic nephropathy , including increased protein synthesis , proliferation , and the expression of tgf-. in these studies , up to 72  h culture period , high glucose did not have a significant effect on viability [ 3235 ] . in order to study
whether high glucose influences cell cycling , 30  mm d - glucose treatment was used for 72  h. in order to control the osmotic effects of high glucose , the normal culture media containing 5.5  mm d - glucose was supplemented with 24.5  mm mannitol .
the results of cell cycle analysis show that both metformin and rapamycin increased the percentage of cells in the g0/g1 phase ( figure 2(a ) ) . in comparison , the effect of metformin on cell cycle inhibition was higher than that of rapamycin . in the high glucose treated conditions , however , metformin - induced cell cycle arrest was abrogated .
in contrast , high glucose did not have an effect on rapamycin - induced cell cycle arrest .
the probability that osmotic effects played a role in the reversal of metformin - induced cell cycle arrest by high glucose is low , as mannitol at an equimolar concentration to high glucose , did not influence cell cycling in the same way .
nevertheless , mannitol treatment caused a significant decrease in the g2/m phase in the metformin - treated condition .
since apoptosis can be induced through g0/g1 arrest , it was important to test whether cell cycle arrest mediated by metformin and rapamycin could have been associated with increased apoptosis . to investigate this ,
the results indicate that neither metformin nor rapamycin caused a statistically significant increase in the percentage of pre - g0/g1 cells ( figure 2(b ) ) .
hek293 cells express both the 1 and 2 isoforms of ampk ; however , it is the 2 subunit that is primarily involved in ampk activation induced by reduced atp synthesis . according to a widely accepted view ,
the cellular effects of metformin involve the inhibition of the mitochondrial electron transport chain ; therefore , it was expected that metformin would exert its effects in an ampk2-dependent manner [ 21 , 42 ] . in order to investigate the involvement of ampk in cell cycle regulation ,
the alpha2 isoform of ampk was knocked down in hek293 cells by a shrna - mediated approach .
ampk2 knockdown was validated by measuring phosphorylation of threonine 172 on both isoforms ( 1 , 2 ) of the alpha subunit ( figures 3(a ) and 3(b ) ) .
in contrast to the nonsilencing condition , in ampk2 knockdown cells metformin did not induce cell cycle arrest in the g0/g1 phase ( figures 3(c ) and 3(d ) ) .
ampk2 deficiency had a comparable effect to high glucose treatment in reversing metformin - induced cell cycle arrest .
relative to cycling cells , g0/g1 arrested cells have a reduced cell size phenotype . in order to investigate
if cell size measurements correlate with the cell cycle experiments , the size of hek293 cells was measured by flow cytometry .
this cell type has been reported to have an intrinsic variation in cell size at various stages of confluence .
nevertheless , in our study no significant differences were detected in the size of hek293 cells up to 60% confluence level ( data not shown ) .
metformin caused approximately 20% decrease in cell size , which was at a comparable level to the effect of rapamycin ( figure 4(a ) ) .
in the combination conditions with high glucose , the effects of the drugs on cell size were reversed , with a more pronounced effect in the metformin - treated condition .
the dependence of metformin on ampk in cell size regulation was also investigated . in line with the results presented in figure 4(a )
, metformin caused a decrease in cell size in the control , nonsilencing condition ( figure 4(b ) ) .
in ampk2-deficient cells metformin inhibited cell size to the same extent as it did in control cells .
as expected , high glucose reversed the effect of metformin on cell size in both control and ampk2-deficient cells .
the role of ampk was further studied by using compound c. some reports have suggested that compound c may promote cell death through apoptosis [ 44 , 45 ] . to investigate the toxic effects of compound c ,
although the viability of cells was not significantly affected by the treatments , cell size was measured on the propidium iodide - negative cell population .
compound c reversed metformin - induced cell size reduction in a concentration - dependent manner , with a maximal effect of compound c at 20  m ( figure 4(c ) ) . in order to investigate whether the effect of compound c on cell size could be associated with corresponding changes in cell cycle ,
cell cycle analysis was done on cells exposed to the same experimental conditions described in figure 4(c ) . at 20  m
, compound c blunted the effect of metformin on inducing an increase in the g0/g1 phase of the cell cycle ( figure 4(d ) ) .
an increased cell size can correlate with increased protein content . as an index of high glucose - induced hypertrophy
the results show that high glucose caused a 25% increase in total protein synthesis over a culture period of two days ( figure 5(a ) ) .
in contrast , metformin did not exert an observable inhibitory effect on total protein synthesis in high glucose - treated cells .
the inhibitory effects of rapamycin and metformin on mtor signalling were assessed by measuring the phosphorylation level of mtor and s6k at ser2448 and thr389 , respectively ( figure 5(b ) ) .
incubating hek293 cells with 8  mm metformin and 100  nm rapamycin for 24  h reduced mtor and s6k phosphorylation , with more pronounced effects on s6k .
in contrast to the combination condition with rapamycin , 30  mm d - glucose pretreatment had an opposing effect on mtor inhibition by metformin . in order to investigate whether high glucose opposes metformin - induced mtor inhibition via ampk , as a marker of its activation , we measured the phosphorylation level of threonine 172 on both isoforms ( 1 , 2 ) of the alpha subunit .
as expected , ampk activation resulted in a concomitant decrease in s6k phosphorylation ( figure 5(c ) ) . in the range of 1530 
mm , d - glucose inhibited ampk phosphorylation and at 25  mm concentration d - glucose treatment opposed the effect of metformin on ampk / s6k signalling .
the expression of p21 was investigated in the context of ampk / mtor / s6k signalling by western blotting .
metformin treatment caused a decrease in p21 expression in a dose - dependent manner ( figure 6(a ) ) .
as expected , metformin caused downregulation of both p - s6k and cyclin d1 with more pronounced effects in the 58  mm concentration range .
high glucose treatments ( 1530  mm ) increased the expression of p21 , cyclin d1 , and p - s6k and at 25  mm concentration blunted the inhibitory effects of metformin .
similar to high glucose , compound c had an opposing effect of metformin - induced ampk activation , s6k dephosphorylation , and p21 downregulation ( figure 6(b ) ) .
the inhibitory effect of metformin on p21 expression was also confirmed in hek293 cells stably expressing shrna against ampk2 ( figure 6(c ) ) . in ampk2-deficient cells ,
metformin - induced ampk activation was reduced . correspondingly , reciprocal changes in mtor phosphorylation could also be observed .
in contrast , in the ampk2 knockdown condition the inhibitory effect of metformin on p21 expression was less pronounced .
the expression of p21 can be regulated by the proteasome and recently it has been suggested that ampk activation may inhibit the function of the proteasome [ 4648 ] . to investigate whether metformin - induced downregulation of p21 is proteasome - dependent ,
the proteasome inhibitor , carbobenzoxy - leu - leu - leucinal ( mg132 ) , was used in control and ampk2-deficient cells . in both control and knockdown cells ,
the downregulation of p21 was prevented by mg132 treatments ( figure 6(d ) ) . in order to confirm the above western blot results and obtain information about changes in p21 localisation
, immunofluorescence microscopy was performed on metformin and high glucose treated hek293 cells . as expected
, metformin treatment decreased the expression of p21 and high glucose treatment reversed metformin - induced p21 downregulation ( figure 7 ) .
in addition , high glucose treatment enhanced the nuclear compartmentalisation of p21 . mtor plays a central role in cell cycle regulation .
furthermore , rapamycin downregulates p21 in mouse fibroblasts . in order to investigate whether mtor inhibition plays a role in p21 regulation , the effect of rapamycin on p21 expression
the inhibition of the mtor signalling pathway was demonstrated by the reduced level of s6k phosphorylation .
this finding was also confirmed in conditionally immortalised human podocytes that expressed p21 in a metformin - sensitive but rapamycin - insensitive manner ( figure 9 ) .
we have found that in hek293 cells high glucose opposes the negative effects of metformin and rapamycin on proliferation , cell size , and protein synthesis , parameters that are widely associated with structural changes early in the development of dn .
it has also been observed that high glucose differentially affects mtor - related phenotypes induced by metformin and rapamycin .
contrary to our expectations , we have found that metformin inhibits p21 expression in a concentration - dependent manner , independently of its effect on mtor signalling .
high glucose , ampk2-deficiency , and compound c overcome the inhibitory effect of metformin on p21 expression , suggesting that ampk may play a role in the regulation of this cyclin - dependent kinase inhibitor .
early cellular changes in the development of dn involve hyperplasia and hypertrophy in both the tubular and mesangial compartments .
these cellular changes have been attributed to increased cellular glucose uptake in cells that are not protected from high ambient glucose levels [ 52 , 53 ] . in the past
decades a considerable interest has been devoted to cell cycle regulatory proteins in glomerular hypertrophy [ 5457 ] .
these studies have suggested that the initial pathological changes in the diabetic kidney are associated with low - grade proliferation followed by cellular hypertrophy . in vitro and in vivo studies
have shown that the absence of p21 prevents the development of hypertrophy associated with the diabetic kidney [ 56 , 58 ] .
it has been shown that the expression of p21 gene ( cdkn1a ) is robustly induced in both type 1 and type 2 diabetic animals . according to a widely held view
however , the role of p21 as a cell cycle inhibitor has been challenged by the finding that p21 may serve as an assembly factor for cyclin d - cdk4 complex formation .
the cyclin d - cdk4 complex is required for cell cycle progression and is known to be ubiquitous among different cell types [ 62 , 63 ] .
furthermore , several studies have suggested that increased cyclin d1 expression is associated with renal and cardiac hypertrophy , which may be attributed to increased stabilisation of p21 [ 6466 ] . in this study ,
high glucose opposed the inhibitory effects of metformin on both cyclin d1 and p21 expression .
in addition , these effects of high glucose correlated with the promotion of cell cycle progression in metformin treated cells , suggesting that high glucose - induced expression of cyclin d1 and p21 may be linked by a common mechanism .
the results of this study suggest that inhibition of ampk may be the underlying mechanism by which high glucose induces p21 expression , which in turn may stimulate proliferation and cell growth .
the involvement of ampk in the regulation of proliferation has been reported in other studies [ 26 , 27 , 67 ] .
in contrast to the 1 isoform , ampk2 has been shown to respond to stress - related conditions , such as hypoxia and glucose deprivation .
furthermore , in ampk2 knockout mice the antihypertrophic effects of metformin are known to be attenuated .
these findings suggest that energy - depleting agents , such as metformin or aicar , exert their antihypertrophic effects through activation of this isoform . however , both drugs may have some limitations in the interpretation of their cellular actions .
it has been reported that , in a time- and concentration - dependent manner , aicar may act as an atp analogue due to the increase in ztp , its triply phosphorylated form . furthermore , it has been suggested that aicar may also mediate ampk - independent processes by regulating amp - sensitive enzymes , such as glycogen phosphorylase .
amp - independent activation of ampk by metformin has also been reported [ 72 , 73 ] .
however , a recent study has demonstrated that aicar - induced ampk activation downregulates p21 expression in retinoblastoma cells .
similarly , despite inhibition of cell cycling , aicar treatment reduced p21 levels in myoblast cultures .
the results of this latter study also show that inhibition of ampk with compound c associates with the reversal of aicar - induced loss of p21 expression .
our finding that compound c abrogates metformin - induced cell cycle arrest is in line with the concept that ampk inhibition promotes proliferation .
furthermore , in experiments studying metformin - induced cellular mechanisms , compound c has been successfully used for blunting ampk activation [ 76 , 77 ] . in accordance with these studies ,
our results also show that compound c opposes the effects of metformin on the ampk / s6k pathway and p21 expression .
evidence suggests that metformin may lower the risk of cancer in patients with diabetes . in cancer studies
the antiproliferative effect of metformin has been attributed to its ability to induce cell cycle arrest through an ampk - dependent mechanism [ 17 , 27 , 29 ] . according to a suggested mechanism , through the activation of ampk , metformin downregulates cyclin d1 , leading to the release of sequestered cdk inhibitors , p27 and p21 . in turn
p27 and p21 may associate with e / cdk2 complexes and inhibit cell cycling at the g1/s checkpoint . on the other hand , in melanoma cells p21 expression
our results show that both metformin and rapamycin inhibit proliferation in hek293 cells by inducing cell cycle arrest in the g0/g1 phase .
interestingly , high glucose pretreatment abrogated the inhibitory effect of metformin on cell cycling but did not affect rapamycin - induced cell cycle inhibition .
these findings suggest that increased p21 expression is not required for cell cycle inhibition by metformin and rapamycin .
the involvement of cell cycle regulatory proteins in the development of dn has long been suggested [ 5 , 56 , 8082 ] .
these studies have suggested that glomerular hypertrophy is a consequence of increased cyclin - dependent kinase inhibitor expression - mediated cell cycle arrest .
the expression of p21 is generally associated with regulation of cell cycle , apoptosis , and ameliorating dna damage . in relation to the ampk / mtor signalling pathway , the induction of p21 as a senescence marker has been described with relevance to age - related diseases .
accumulating evidence indicates that senescence may play an important role in the development of dn .
premature senescence has been observed in fibroblasts and proximal tubular cells isolated from patients with dn [ 8587 ] .
downregulation of connexin 43 , a gap junction protein , has been reported in podocytes of diabetic patients as well as in high glucose treated glomerular mesangial cells that showed increased expression of senescence markers , such as p21 , p27 , and -galactosidase staining [ 82 , 88 ] .
furthermore , high glucose induces cyclin d1 expression , and interestingly , increased cyclin d1 expression is associated with senescence in fibroblasts .
diabetes contributes to vascular ageing , and endothelial cell senescence is induced by high glucose or advanced glycosylated end products . in a study on rat kidney
proximal tubule cells , an increase in the expression of p21 and p27 was associated with a phenotypic transition to senescence .
however , it has been shown that dedifferentiation of cells in the kidney may contribute significantly to the pool of proliferating cells [ 92 , 93 ] . in turn , dedifferentiated cells responding to mitogenic factors can progress to senescent arrest .
overexpression of fibronectin , one of the extracellular matrix proteins induced in diabetic nephropathy , is one of the characteristics of senescence cells .
furthermore , proteasomal protein degradation is reduced in senescent cells , which can be a contributing factor for increased hypertrophy .
the involvement of apoptosis in diabetic nephropathy has been indicated by several investigators [ 9698 ] .
it has been widely acknowledged that apart from senescence , damaged cells rely on apoptosis to evade tumour formation .
it has been suggested that apoptotic cell loss may be a process of normal tissue homeostasis regulating mesangial cell populations in enlarged glomeruli [ 100 , 101 ] .
have demonstrated using diabetic rats that apoptosis may differentially affect glomerular cells in small and large glomeruli .
they also showed that cellular hypertrophy was responsible for the differences in size , as the expression level of fibronectin , an extracellular matrix marker , was the same in both small and large glomeruli .
increased expression of proapoptotic proteins was found in large glomeruli , suggesting that apoptosis may selectively operate in a more hypertrophied cellular environment .
interestingly , upregulation of cyclin d1 , p21 , and p27 was only detected in smaller glomeruli , indicating that the higher expression of these proteins is associated with the initiation of glomerular hypertrophy .
inhibition of mtor by rapamycin has been found to prevent the permanent loss of proliferative potential that is characteristic of cellular senescence .
since metformin also inhibits the mtor signalling pathway , a similar effect of metformin to that of rapamycin could be expected in the prevention of senescent transformation . however ,
in contrast to rapamycin , chronic exposure to lower doses of metformin has been shown to promote cancer - protective senescence in other studies [ 40 , 103 ] .
it has been proposed that rapamycin regulates proliferation by preventing the formation of cyclin - dependent kinase complexes .
mtor activation by growth factors or high energy levels induces p21 that in turn facilitates the assembly of these complexes . in this study metformin
inhibited p21 in both hek293 cells and human podocytes , suggesting that energy depletion may affect the expression of this cell cycle inhibitor .
the underlying factor that determines the cellular actions of ampk may be its level of activation [ 84 , 105 ] .
however , a major limitation to understanding the role of ampk in the diabetic kidney is the lack of studies investigating cell - specific differences in ampk expression .
cells in the medulla predominantly depend on glycolytic metabolism , whereas tubular cells in the cortex depend on oxidative metabolism [ 107 , 108 ] .
glycolytic flux is known to be reduced during the process of cellular senescence ; therefore one can envisage that senescence - induced metabolic disturbances may differentially impact on cell populations in the kidney [ 109 , 110 ] .
immortalised cells may have a relatively high glycolytic flux ; therefore , it is possible that in our experiments high glucose exerted its effects through enhanced glycolysis . in preclinical studies ,
the doses of metformin are much higher than the level of the drug reported to accumulate in tissues after oral administration ; therefore , it is difficult to extrapolate the results of these studies to a clinical setting because of the differential and possibly poor expression of octs in immortalised cell lines .
nevertheless , these studies show that metformin may have beneficial effects in pathologic states , including cancer and inflammation [ 17 , 112 , 113 ] .
several molecular pathways in these diseases are also relevant to the aetiology of diabetic nephropathy , suggesting that metformin may have a therapeutic potential for the prevention of dn .
indeed , by modulating the expression of proinflammatory genes , metformin has been shown to ameliorate dn in rats and ampk activation by metformin reduced renal hypertrophy in diabetic rats [ 114 , 115 ] .
in summary , the above indicates that metformin can reduce the expression of p21 and ampk may play a role in the underlying mechanism .
it can also be inferred from these results that p21 is not required for metformin - induced cell cycle arrest .
this finding lends support to other studies that look beyond the role of p21 as a cell cycle inhibitor [ 74 , 75 ] .
metformin has implications for the treatment of both diabetes and cancer and p21 has different , poorly understood roles in both diseases .
recently , energy sensing pathways have been investigated in the context of both diabetic complications and cancer [ 116118 ] .
in addition , reentry of differentiated cells into the cell cycle as well as genetic polymorphism in the p21 gene has been implicated in alzheimer 's disease [ 119 , 120 ] .
 the mammalian target of rapamycin ( mtor ) pathway plays an important role in the development of diabetic nephropathy and other age - related diseases . 
 one of the features of dn is the elevated expression of p21waf1/cip1 . 
 however , the importance of the mtor signalling pathway in p21 regulation is poorly understood . 
 here we investigated the effect of metformin and rapamycin on mtor - related phenotypes in cell lines of epithelial origin . 
 this study reports that metformin inhibits high glucose - induced p21 expression . 
 high glucose opposed metformin in regulating cell size , proliferation , and protein synthesis . 
 these effects were associated with reduced ampk activation , affecting downstream mtor signalling . 
 however , the inhibition of the mtor pathway by rapamycin did not have a negative effect on p21 expression , suggesting that metformin regulates p21 upstream of mtor . 
 these findings provide support for the hypothesis that ampk activation may regulate p21 expression , which may have implications for diabetic nephropathy and other age - related pathologies . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion