hyperinsulinemia and elevated circulation angiotensin ii ( ang ii ) level tend to concomitantly occur in obesity patients and contribute to obesity - related hypertension . in recent years
, a number of studies found a cross talk , at multiple levels , between ang ii and insulin [ 26 ] .
several studies showed that ang ii could negatively modulate insulin - mediated actions [ 24 ] . at the intracellular level
, ang ii was found to work on jak-2/irs1-irs2/pi3 kinase , jnk , and erk via ang ii receptor type 1 ( at1r ) , to phosphorylate serine residues of key components of insulin signaling pathway , that is , the insulin receptors , irs1 , and the p85 subunit of pi3 kinase , thereby inhibiting pi3 kinase / akt signaling pathway .
in addition , by inducing expression of the regulatory protein socs 3 , ang ii may inhibit insulin - induced tyrosine phosphorylation of irs1 and irs2 and [ ser473 ] phosphorylation of akt , as a consequence , impairing the transduction of insulin signals in the jak2/stat-5b pathway [ 5 , 6 ] .
it is generally believed that ang ii acts on insulin predominantly by inhibiting pi3 kinase / akt pathway .
nonetheless , researchers do not entirely agree about the role of the map kinase pathway in the cross talk between ang ii and insulin .
mayer and colleagues found that , after treatment with ang ii , the phospho - erk1/2 activity at the hypothalamic level was significantly higher in rats pretreated with insulin than in those treated with insulin or ang ii alone .
studies by carvalheira et al . also suggested a direct and positive cross talk between ang ii and insulin in erk pathway in cardiac tissues . on the other hand
, other studies revealed a competitive cross talk between ang ii and insulin - mediated erk pathways . in the epithelial cells of renal proximal tubules ,
insulin - mediated erk activation was found to be suppressed by ang ii , although the two hormones , when working separately , augmented erk1/2-type kinase activity . similarly , a study observed that , in at1ar - ok cells ( ok cells that stably express transfected at1ar ) , insulin suppressed ang ii - mediated erk phosphorylation . the cross talk between ang ii and insulin has been described mainly in cardiovascular cells , hepatocytes , adipocytes , skeletal muscles , and so forth , and , so far , no such cross talk was reported in adrenal .
ang ii , insulin , and insulin - like growth factor 1 ( igf-1 ) were shown to play important roles in adrenocortical cells [ 1113 ] , and overexpression of igf-1 receptor was found to be associated with the development of adrenocortical carcinoma [ 1417 ] . in this study , by employing adrenocortical carcinoma h295r cells [ 1822 ] , we examined the interaction between ang ii and insulin / igf-1 in erk and akt signaling pathways and expression of steroidogenic enzymes in the cells .
human recombinant ang ii , insulin , igf-1 , and at2 receptor blocker pd123319 were procured from sigma - aldrich ( st . louis , mo , usa ) .
pkc inhibitor g6983 , pi3k inhibitor ly294202 , and mek1/2 inhibitor u0126 were purchased from calbiochem ( san diego , ca , usa ) .
at1 receptor blocker candesartan was from astrazeneca . neutralizing anti - igfr1 antibody , mab 391 , was bought from r & d systems ( minneapolis , mn , usa ) .
dulbecco 's modified eagle 's medium / f12 ( dmem / f12 ) was from life technologies ( carlsbad , ca , usa ) .
nu serumtm and its+premix were obtained from bd biosciences ( bedford , ma , usa ) .
anti - phospho - erk1/2 ( thr202/tyr204 ) , anti - phospho - akt ( ser473 ) , anti - erk1/2 , and anti - akt antibodies were from cell signaling technology ( danvers , ma , usa ) .
secondary antibodies [ irdye 800cw conjugated goat ( polyclonal ) anti - mouse igg and irdye 680 conjugated goat ( polyclonal ) anti - rabbit igg ] were products of li - cor biosciences ( lincoln , ne , usa ) .
nci - h295r cells , a human adrenocortical cell line nci - h295r ( atcc , rocksville , md , usa ) , were cultured in dmem / f12 , containing 2.5% nu serum , 1% its plus premix [ containing insulin ( 6.25  g / ml ) , transferrin ( 6.25  g / ml ) , selenium ( 6.25  ng / ml ) , bsa ( 1.25  mg / ml ) , and linoleic acid ( 5.35  g / ml ) ] , 50 units / ml penicillin , and 50  g / ml streptomycin , at 37c in an incubator supplied with humidified air
. the cells were passaged by digestion with trypsin - edta ( 0.25% trypsin and 0.02% edta in pbs without ca and mg ) . for each experiment ,
the cells were cultured in six - well plates with the aforementioned growth medium . at 6070% confluence of the cells ,
the culture medium was replaced by serum - free dmem / f12 at 37c for 24  h before specific stimulation .
after the stimulation for indicated times , the media were aspirated , and cells were washed three times with ice - cold pbs and then lysed in 100  l of laemmli sample buffer .
after sonication , centrifugation , and heating at 95c for 5  min , the supernatant proteins were analyzed on sds - page ( 616% ) gradient gels and transferred to polyvinylidene difluoride membranes .
blots were blocked for 1  h in 5% fat - free milk and incubated overnight at 4c with primary antibodies .
after washing three times with pbs containing 0.1% tween 20 for 5  min , blots were incubated with secondary antibodies at room temperature , with gentle shaking , for 1  h , and then washed three times with pbs containing 0.1% tween 20 .
immunoblots were densitometrically analyzed on an odyssey infrared imager ( li - cor biosciences , lincoln , usa ) . cells were cultured in 24-well plates with growth medium for 1 to 2 days until they became subconfluent . then the medium was replaced with serum - free dmem / f12 and the cells were treated with different agonists .
after 24 hours , the cells were lysed with trizol reagent , and total rna was isolated .
cdna was synthesized from 1  g total rna by using a high - capacity cdna reverse transcription kit .
cyp11b1 and cyp11b2 genes were pcr amplified by using the lightcycler 480 sybr green i master on a lightcycler 480 pcr system .
the relative expression of each gene was quantitatively determined by normalizing against the expression of gapdh gene .
the primers used were as follows : cyp11b1 , 5-aatgcggaactgtcgccagatg-3 ( forward ) and 5-tcagcaagggaaacaccgtc-3 ( reverse ) ; cyp11b2 , 5-actcgctgggtcgcaatg-3 ( forward ) and 5-agtgtctccaccaggaagtgc-3 ( reverse ) ; and gapdh , 5-cggagtcaacggatttggtc-3 ( forward ) and 5-tgggtggaatcatattggaacat-3 ( reverse ) .
all values were expressed as mean  sd , and anova with a post hoc test was conducted for data comparison between groups by using spss software package ( version 13.0 ) .
stimulation of h295r cells with ang ii for 5  min caused a dose - dependent phosphorylation of erk1/2 , with the maximum effect occurring at the concentration of 100  nm and 1000  nm ( figure 1 ) .
both 100  nm insulin and 1000  nm insulin could induce akt phosphorylation whereas only at the highest dose ( 10  nm ) igf-1 induced akt phosphorylation ( figures 2(a ) and 2(b ) ) .
compared to the control , 100  nm ang ii increased phospho - erk1/2 approximately 3-fold .
insulin ( 100  nm ) or igf-1 ( 10  nm ) alone raised phospho - erk1/2 1.8- and 1.5-fold , respectively , while , after pretreatment with 100  nm ang ii for 30  min , insulin ( 100  nm ) or igf-1 ( 10  nm ) elevated phospho - erk1/2 level 8- and 7-fold , respectively ( figures 2(c ) and 2(d ) ) .
phospho - erk1/2 was increased much more by ang ii in combination with insulin / igf-1 than by ang ii or insulin / igf-1 alone , indicating that the elevation was caused by more than an additive effect .
ang ii marginally suppressed akt activation under the basal condition , while it exerted no effect on phospho - akt induced by insulin / igf-1 ( figures 2(c ) and 2(d ) ) .
the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation was abolished by candesartan , a selective at1 receptor blocker , but not by pd1233319 , a selective at2 receptor blocker , indicating that the action of ang ii was mediated by at1 receptor activation .
mab 391 , a neutralizing anti - igf-1 receptor antibody , significantly attenuated ang ii- and igf-1-induced phosphorylation of erk1/2 and akt , while it had no effect on the ang ii- and insulin - induced responses ( figure 3 ) .
ang ii is known to stimulate pkc and mek pathway , while insulin / igf-1 plays its role mainly through pi3k pathway . in h295r cells , both pkc inhibitor and mek1/2 inhibitor
fully inhibited ang ii - stimulated phospho - erk1/2 and substantially decreased phospho - erk1/2 stimulated by ang ii plus insulin / igf-1 .
both erk1/2 inhibitor u0126 and pkc inhibitor g6983 showed a marginal stimulatory effect on phospho - akt , under basal condition or after treatment with agonists ( figures 4 and 5 ) .
pi3k inhibitor altogether abolished insulin / igf-1 stimulated phospho - akt and markedly decreased phospho - akt costimulated by ang ii and insulin / igf-1 ( figure 6 ) . to clarify whether the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation affects the cellular function , we observed mrna expression of the steroidogenic enzymes stimulated by ang ii and insulin / igf-1 , alone or combined .
as shown in figure 7 , ang ii significantly stimulated mrna expression of cyp11b1 and cyp11b2 .
insulin receptor ( ir ) and type 1 igf receptor ( igf-1r ) serve distinctly different functions . they both possess a highly conserved structure , with a 60% homology between them .
each receptor has a homodimeric structure consisting of two  and two  chains covalently linked in the membrane .
these two receptors may engage in synthesis of a hybrid receptor ( ir - igf-1r heterodimers ) .
monoclonal antibodies against igf-1r were found to decrease the cell surface expression of igf-1r homodimers and ir - igf-1r heterodimers by causing receptor internalization .
the endogenous ligands of ir include insulin , igf-1 , and igf-2 , but igf-1r is activated only by igf-1 and igf-2 . in this study
, we found that mab 391 , a monoclonal antibody of igf-1r , significantly suppressed phosphorylation of erk1/2 and akt costimulated by ang ii and igf-1 in h295r cells , suggesting that igf-1 exerted its effect predominantly through igf-1r in adrenocortical cells . mab 391 had no effect on the responses induced by ang ii plus insulin , suggesting that insulin acts mainly on ir , and no or hardly any ir - igf-1r heterodimers are present in these cells .
although the roles of insulin and igf-1 in adrenocortical cells are well documented , only two studies examined intracellular erk1/2 and akt pathways [ 25 , 26 ] .
they reported that igf-1 activated the phosphorylation of both erk1/2 and akt [ 25 , 26 ] .
the present study showed that insulin and igf-1 exerted a weak stimulatory effect on erk phosphorylation and a strong stimulatory impact on akt phosphorylation .
in adrenocortical cells , after activation of ir and igf-1r , their effects on erk1/2 and akt signaling pathway are similar .
we found that , in h295r cells , ang ii suppressed akt activation under the basal condition , while it had no effect on phospho - akt induced by insulin / igf-1 .
insulin signaling was found to be inhibited by ang ii in several other cells [ 27 , 28 ] . in vascular smooth muscle cells ( vsmc ) ,
ang ii - elicited erk1/2 activation leads to phosphorylation of irs1 at ser307 and ser616 , thereby inhibiting akt activation and ultimately suppressing the insulin - induced glucose uptake .
reported that ang ii inhibited insulin - mediated glut4 translocation in rat skeletal muscle cells through at least two pathways : ( 1 ) the transient activation of erk1/2 which inhibits irs1/2 and ( 2 ) direct inhibitory nitration of akt .
the major findings of the present study were that ang ii and insulin / igf-1 exerted a synergistic stimulatory effect on erk1/2 activation in adrenocortical cells .
only two in vivo studies showed additive stimulatory effect of these two hormones on erk1/2 activation in hypothalamus and heart tissues [ 7 , 8 ] .
activation of erk1/2 by ang ii through at1 receptor may involve three different pathways , that is , gq - pkc , src - ras , and transactivation of the epidermal growth factor receptor , while erk1/2 activation by insulin was found to be mainly through shc - grb2-ras pathway [ 29 , 30 ] . as shown in this study , in adrenocortical cells , pkc was the major pathway involved in ang ii - induced erk1/2 activation .
the mechanism underlying the synergistic stimulation is still unknown and might involve other unknown factors .
we believe that this synergistic effect can not be explained simply by additive effect of the two hormones .
ang ii could stimulate aldosterone and cortisol secretion , which might be mediated by erk1/2 activation [ 31 , 32 ] .
we observed that the stimulatory effects of ang ii on the expression of steroidogenic enzymes could be enhanced by cotreatment with insulin / igf-1 .
the results indicated that the synergistic stimulatory effect of ang ii and insulin / igf-1 on erk1/2 activation might be involved in the enhanced steroid hormone production stimulated by ang ii plus insulin / igf-1 .
this study , for the first time , demonstrated that the interaction between ang ii and insulin / igf-1 was evidently of synergistic , rather than additive , nature in stimulating erk1/2 activation in h295r cells . such synergistic stimulatory effect on erk1/2
 the cross talk between angiotensin ii ( ang ii ) and insulin has been described mainly in cardiovascular cells , hepatocytes , adipocytes , and so forth , and to date no such cross talk was reported in adrenal . in this study , we examined the interaction between ang ii and insulin / igf-1 in erk and akt signaling pathways and expression of steroidogenic enzymes in h295r cells . 
 compared to the control , 100  nm ang ii increased phospho - erk1/2 approximately 3-fold . 
 insulin ( 100  nm ) or igf-1 ( 10  nm ) alone raised phospho - erk1/2 1.8- and 1.5-fold , respectively , while , after pretreatment with 100  nm ang ii for 30  min , insulin ( 100  nm ) or igf-1 ( 10  nm ) elevated phospho - erk1/2 level 8- and 7-fold , respectively . 
 the synergistic effect of ang ii and insulin / igf-1 on erk1/2 activation was inhibited by selective at1 receptor blocker , pkc inhibitor , and mek1/2 inhibitor . 
 ang ii marginally suppressed akt activation under the basal condition , while it had no effect on phospho - akt induced by insulin / igf-1 . 
 ang ii significantly stimulated mrna expression of cyp11b1 and cyp11b2 , and such stimulatory effects were enhanced when cells were cotreated with insulin / igf-1 . 
 we are led to conclude that ang ii in combination with insulin / igf-1 had an evident synergistic stimulatory effect on erk1/2 activation in h295r cells and the effect may be responsible for the enhanced steroid hormone production induced by ang ii plus insulin / igf-1 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions