Patent Application: US-39886906-A

Abstract:
the invention relates to new methods and compositions for treating diabetics , pre - diabetics , and patients at risk of becoming diabetic or with impaired glucose tolerance . the invention , in one embodiment , involves activating insulin receptor substrate - 2 to protect against loss of beta cell mass , protect against loss of beta cell function , rejuvenate beta cells mass , rejuvenate beta cell function or any combination thereof , thereby stimulate insulin production using an effective amount of lys b3 , glu b29 insulin to patients in need of this treatment .

Description:
the potentially enhanced mitogenic activity of insulin analogs may affect the safety profile of the human hormone and requires a detailed analysis of any new analog considered for therapeutic applications . the signaling properties and the mitogenic potency of two novel rapid acting insulin analogs , lys b3 , glu b29 insulin ( hmr 1964 ) and lys b3 , ile b28 insulin ( hmr 1153 ) have been assessed in comparison to native human insulin and the analog asp b10 insulin using rat and human myoblasts and differentiated muscle cells . in k6 myoblasts expressing a high level of igf - i receptors , both binding and internalization were 2 - 3fold higher for asp b10 insulin and hmr 1153 when compared to hmr 1964 and regular insulin . this correlated with a prominent shc / igf - i receptor interaction , tyrosine phosphorylation of shc , activation of erk1 and erk2 , and stimulation of dna synthesis by hmr 1153 and asp b10 insulin . in contrast , hmr 1964 produced a marginal activation of the shc / map kinase cascade and was equipotent to insulin in stimulating dna synthesis in k6 myoblasts . in these cells hmr 1964 produced a minor activation of irs - 1 tyrosine phosphorylation despite a significantly higher autophosphorylation of the igf - i receptor when compared to insulin . however , this analog produces a prominent activation of irs - 2 with a significantly stronger effect than insulin in human myoblasts . preferential activation of irs - 2 was also observed in differentiated cardiomyocytes where hmr 1964 increased 3 - o - methylglucose transport to the same extent as human insulin . thus i ) the mitogenic properties of insulin analogs may result from a complex series of initial receptor interactions including internalization and phosphorylation , ii ) the primary structure of the insulin molecule may be sufficient to control hormonal action at the downstream level , and iii ) the mitogenic potential of hmr 1964 is identical to that of insulin and that selective activation of irs - 2 by this analog may open new avenues for optimized insulin therapy . for example , irs - 2 activation by hmr 1964 may be a way of protecting against loss of beta cell mass , protecting against loss of beta cell function , rejuvinating beta cells mass or rejuvinating beta cell function or any combination thereof , which in returns leads to insulin production . in one embodiment , a protective or rejuvinatory effect to beta cells will not likely be observed upon a first administration of hmr 1964 . one of skill in the art will recognize that the amount of time necessary will depend on the extent of beta cell damage present and the amount of hmr 1964 administed . for example , regular administration for one week or more , such as two weeks or three weeks may be necessary , as may administration for one month or greater , such as , for example , two months , three months or greater . the amount of hmr 1964 administed will of course depend on the disease state being treated and / or the amount of damage to beta cell function and mass . for example , in one embodiment , hmr 1964 is administered in an amount effective to protect against loss of beta cell mass , protect against loss of beta cell function , rejuvenate beta cells mass or rejuvenate beta cell function or any combination thereof without corresponding signficant reduction in blood glucose levels . corresponding , as used in this context , refers to a signficant reduction in blood glucose levels that occurs after administration of hmr 1964 , for example , immediately after , one hour , two hours , three hours , 6 hours , or 12 hours following administration . in one embodiment , hmr 1964 is administered in an amount effective to deliver hmr 1964 to the pancreas but not substantially lower blood glucose levels . as used herein , substantially lowering blood glucose levels refers to a therapeutically effective lowering of blood glucose levels . in another embodiment , hmr 1964 may be administered in an amount effective to deliver hmr 1964 to the pancreas for a protective or rejuvenatory effect yet still substantially lower blood glucose levels . in one embodiment , the amount administered is at the upper limit below the amount of insulin normally administered to a diabetic . for example , per day , less than about 2 iu / kg , such as , for example , about 1 . 5 iu / kg , about 1 . 0 iu / kg , about 0 . 5 iu / kg , about 0 . 25 iu / kg , and about 0 . 1 iu / kg or less . the lower limit may , for example , in one embodiment , be about 0 . 01 iu / kg or greater , such as , for example , about 0 . 025 iu / kg , about 0 . 05 iu / kg , about 0 . 07 iu / kg , about 0 . 08 iu / kg , and about 0 . 09 iu / kg or greater . low amounts of hmr 1964 may be administed in a combination therapy with other insulins , such as human insulin . thus , in one embodiment , a pharmaceutical composition of the invention is a pharmaceutical composition consisting essentially of hmr 1964 , wherein said hmr 1964 is present in an amount ranging from about 0 . 01 iu / kg to about 0 . 1 iu / kg or a pharmaceutical composition comprising hmr 1964 with the provisio that said pharmaceutical composition does not contain human insulin , wherein said hmr 1964 is present in an amount ranging from about 0 . 01 iu / kg to about 0 . 1 iu / kg . native human insulin and the insulin analogs lys b3 , glu b29 insulin ( hmr 1964 ), lys b3 , ile b28 ( hmr 1153 ) and asp b10 insulin as well as the 125 i - labeled insulin preparations ( specific activity 260 mci / mg ) were provided by aventis pharma gmbh ( frankfurt , germany ). 3 - o -[ 14 c ] methyl - d - glucose and l -[ 1 - 14 c ] glucose were purchased from amersham pharmacia biotech ( freiburg , germany ). reagents for sds - page were supplied by amersham pharmacia biotech and sigma ( deisenhofen , germany ). collagenase was from serva ( heidelberg , germany ) and bovine serum albumin ( bsa , fraction v , fatty acid free ) was obtained from boehringer mannheim ( germany ). protein a - trisacryl ( gf - 2000 ) and protein g - agarose were products from pierce ( oud beijerland , the netherlands ). the monoclonal igf - i receptor antibody was purchased from oncogene research products ( cambridge , mass ., usa ). the polyclonal anti - shc , anti - irs 1 and anti - irs 2 antibodies were obtained from biomol ( hamburg , germany ). irs1 and irs 2 antisera used for immunoprecipitation were kindly provided by dr . j . a . maassen ( leiden , the netherlands ). the phosphospecific p42 / 44 map - kinase antisera ( thr202 / tyr204 ) and the p44 / 42 map - kinase antibodies were products of new england biolabs ( schwalbach / taunus , germany ). the anti - phosphotyrosine antiserum rc20 was produced by becton dickinson ( heidelberg , germany ). horseradish peroxidase conjugate ( anti - rabbit igg ) as the secondary antibody for ecl was purchased from promega ( mannheim , germany ). stripping solution was a product of alpha diagnostics ( san antonio , tex ., usa ). the cell proliferation elisa chemiluminescence kit was purchased from boehringer ( mannheim , germany ). fetal calf serum , dulbecco &# 39 ; s modified eeagle medium ( dmem ), non - essential amino acids and penicillin / streptomycin were provided from gibco ( eggenstein , germany ). primary human skeletal muscle cells , basal medium and supplement pack for growth medium were obtained from promocell ( heidelberg , germany ). all other chemicals were of the highest grade commercially available . k6 myoblasts represent a rat heart muscle cell line that was established and characterized ( 16 ). these cells are insulin - sensitive and express the glucose transporter glut4 ( 16 ). cells were kept in monolayer culture in dmem supplemented with 10 % fetal calf serum , non essential amino acids ( 1 %), streptomycin ( 100 μg / ml ) and penicillin ( 100 u / ml ) in 175 cm 2 flasks in an atmosphere of 5 % co 2 at 37 ° c . myoblasts were maintained in continuous passages by trypsinization of subconfluent cultures 7 days after plating . the medium was changed every 72 h . cell number was determined after cell dissociation with trypsin / edta at 37 ° c . primary human skeletal muscle cells obtained from satellite cells isolated from m . rectus abdominis of a 28 year old male caucasian donor were supplied as proliferating myoblasts . these cells were kept in skeletal muscle cell growth medium ( basal medium containing : fetal calf serum , 5 %; epidermal growth factor , 10 ng / ml ; basic fibroblast growth factor , 1 ng / ml ; fetuin , 0 . 5 mg / ml ; insulin , 0 . 1 mg / ml ; dexamethasone , 0 . 4 μg / ml ; gentamicin , 50 μg / ml ; and amphotericin b , 50 ng / ml ) for two population doublings . cells were then frozen and stored in liquid nitrogen until further use . for stimulation experiments , 10 6 cells / dish were seeded in growth medium and cultured for 2 days . cells were then washed with pbs and cultured for 4 days in the absence of serum and insulin . the cells were then cultured for 1 h with fresh medium containing 0 . 5 % bsa , and subsequently stimulated with the hormones . adult rat cardiomyocytes were isolated by perfusion of the heart with collagenase , as previously described by us ( 17 ). male wistar rats ( 280 - 340 g ) were used in all experiments . the final cell suspension was incubated for 60 min until further use in hepes buffer ( 130 mm nacl , 4 . 7 mm kcl , 1 . 2 mm kh 2 po 4 , 25 mm hepes , 5 mm glucose , 2 % ( w / v ) bovine serum albumin , ph 7 . 4 , equilibrated with oxygen ) containing mgso 4 and cacl 2 ( final concentrations : 1 mm ) at 37 ° c . in a rotating waterbath shaker . the cell viability was judged by determination of the percentage of rod - shaped cells and averaged 90 - 97 % under all incubation conditions . for binding studies myoblasts were suspended in dmem containing 10 % fetal calf serum ( fcs ) and seeded in 6 well culture dishes at a density of 2 × 10 5 cells / well . after 24 h in culture the cells were washed 2 times with phosphate - buffered saline ( pbs ) and incubated for 60 min at 37 ° c . in dmem without fcs containing 2 % bovine serum albumin ( bsa ). 125 i - labeled human insulin or one of the 125 i - labeled insulin analogs ( 0 . 1 μci , 5 × 10 − 11 m ) was then added along with the corresponding unlabeled peptide hormone ( 10 − 8 m ), and incubation was continued for 10 min at 37 ° c . the medium was then removed , the cells were washed twice and lysed with 0 . 1 % sodium dodecylsulfate ( sds ) and the radioactivity was determined in a gamma counter . non specific binding was measured in parallel incubations performed in the presence of an excess of the corresponding unlabeled hormone ( 10 − 5 m ), respectively . all assays were performed in triplicate . internalization was determined after incubation of myoblasts for 60 min at 37 ° c . using a concentration of 5 × 10 − 11 m ( 0 . 1 μci ) of the different insulin molecules . unbound insulin was first removed by washing the cells with cold pbs , followed by washing the cells three times with cold pbs at acid ph ( ph 2 . 75 , 0 . 1 % bsa ). cells were lyzed in 1 % sds / 0 . 1 n naoh , and the remaining radioactivity was determined in a gamma counter . the same incubation conditions were also used for determination of the degradation of the insulin molecules . briefly , after 60 min aliquots of the supernatant were subjected to trichloroacetic acid ( tca ) precipitation and the degradation was calculated from the increase in tca solubility of the tracer , as outlined earlier ( 18 ). k6 myoblasts ( 3 × 10 6 ) were plated in their regular growth medium . after a 24 h culture period the medium was removed and replaced with fresh medium without fcs containing 0 . 5 % bsa . following a 2 h incubation at 37 ° c . cells were stimulated with human insulin or one of the insulin analogs ( final concentration 5 × 10 − 7 m ) for 10 min . after washing twice with ice - cold pbs , cells were incubated in ripa lysis buffer ( 50 mm tris - hcl ( ph 7 . 4 ), 1 % np - 40 , 0 . 25 % na - deoxycholate , 150 mm nacl , 1 mm edta , 1 mm pmsf , 1 μg / ml each aprotinin , leupeptin , pepstatin , 1 mm na 3 vo 4 and 1 mm naf ) for 2 h at 4 ° c . with gentle agitation . human myoblasts were cultured as outlined above and stimulated with the insulin molecules for 10 min followed by lysis with ripa buffer . freshly isolated cardiomyocytes were preincubated in a rotating water - bath shaker according to our protocols ( 19 ), and after a 10 min stimulation with the different insulin analogs the lysis was performed using ripa buffer . for immunoprecipitation cell lysate samples were then incubated with antibodies against the igf - i receptor , irs - 1 , irs - 2 or shc at 4 ° c . and gently rocked overnight . the immunocomplexes were adsorbed on to protein g - sepharose or protein a - sepharose beads for 2 h at 4 ° c . during gentle agitation before being collected by centrifugation at 14 , 000 rpm for 30 s . beads were washed 3 times with ice - cold pbs and used for western blot analysis . the immunoadsorbed proteins were solubilized in laemmli sample buffer and were resolved by sds / page on 8 - 18 % ( w / v ) horizontal gradient gels , followed by transfer to polyvinylidene difluoride ( pvdf ) membranes . these were then blocked in tbs / tween 0 . 05 % plus 1 % bsa for 1 h at room temperature and incubated with the appropriate primary antibody at 4 ° c . overnight . after extensive washing , membranes were incubated with horseradish peroxidase - conjugated secondary antibodies . protein bands were then visualized by the enhanced chemiluminescence ( ecl ) method on a lumilmager work station . map - kinase activation was assessed by immunobloting k6 cell lysates with phosphospecific erk½ antibodies and the phosphorylated proteins were detected by the ecl method . the blots were stripped and reprobed with the polyclonal erk½ antibody as described by the manufacturer . all blots were quantified using the lumilmager software . for monitoring dna synthesis , k6 myoblasts ( 1 × 10 4 cells / well ) were seeded in 24 - well microtiter tissue culture plates and were cultured for 24 h in dmem containing 10 % fcs , followed by a 30 h culture period under serum - free conditions . cells were then stimulated with the different peptide hormones or 10 % fcs for 16 h with the simultaneous addition of 5 - bromo - 2 ′- deoxyuridine ( brdu ). after removing the labeling medium , the cells were fixed and the dna was denatured by addition of fixdenat ( boehringer mannheim , germany ). cells were then incubated with a peroxidase - conjugated anti - brdu antiserum , and after addition of substrate the light emission was quantified on a lumilmager work station . the determination of 3 - o - methylglucose transport using freshly isolated adult rat cardiomyocytes was performed at 37 ° c . in hepes buffer ( composition : 130 mm nacl , 4 . 8 mm kcl , 1 . 2 mm kh 2 po 4 , 1 mm mgcl 2 , 1 mm cacl 2 , 25 mm hepes , 5 mm glucose , 20 g / l bsa , ph 7 . 4 ). 4 × 10 5 cells / ml were stimulated with insulin or insulin analogs for 10 min . the transport reaction was started by pipetting a 50 μl aliquot of the cell suspension to 50 μl of hepes buffer containing 3 - o -[ 14 c ] methyl - d - glucose ( final concentration 100 μm ). carrier - mediated glucose transport was then determined using a 10 - s assay period and l -[ 14 c ] glucose to correct for simple diffusion , as described in earlier reports from this laboratory ( 19 , 20 ). all results are expressed as means ± sem . the significance of reported differences was evaluated by using the null hypothesis and t statistics for paired data . corresponding sigificance levels are indicated in the figures . it has been reported that modifications in the c - terminal region of the b - chain of insulin alter the affinity for the igf - i receptor more than for the insulin receptor ( 21 ). as shown previously ( 13 ), cardiac myoblasts express a high level of igf - i receptors with a marginal abundance of insulin receptors , thus representing a suitable tool to assess igf - i receptor signaling by insulin analogs . as shown in table 2 , the analog hmr 1153 exhibited the highest binding to k6 myoblasts being comparable to the analog asp b10 insulin , which has a reported higher affinity for the igf - i receptor than regular insulin ( 22 ). in contrast the analog hmr 1964 showed a significantly lower binding potency that was similar to human insulin . it should be noted that these binding studies were performed at a high concentration of the peptide hormones ( 10 − 8 mol / l ) in order to allow comparison to the studies on signal transduction and dna synthesis described below . interestingly , hmr 1964 also exhibited the lowest rate of internalization and low degradation by the myoblasts ( table 2 ). the data in table 2 also show that internalization and degradation of an insulin molecule are not always directly correlated . thus , human insulin showed a significantly lower rate of internalization compared to hmr 1153 , but showed the highest degradation by k6 cells . this fits to the notion that insulin processing reflects a complex process involving internalization and degradation at different intracellular sites ( 23 ). in order to assess the signaling potency of the different insulin analogs to the map - kinase pathway , the autophosphorylation of the igf - i receptor , the phosphorylation of shc proteins and the activation of p44 / 42 ( erk½ ) map - kinase was determined . as presented in fig1 , the analog hmr 1153 induced a very prominent autophosphorylation of the igf - i receptor in the k6 myoblasts . this effect was about 2 . 5 fold higher than the autophosphorylation induced by asp b10 insulin despite a comparable binding of these analogs ( see table 2 ). on the other hand , the analog hmr 1964 produced the same effect as asp b10 insulin despite the lowest binding affinity to the k6 myoblasts . thus , the level of receptor occupancy may not be sufficient to determine the signaling potency of an insulin molecule . this is also shown in fig2 . immunoprecipitates of the igf - i receptor were immunoblotted with an anti - shc - antibody that recognizes all three shc - isoforms . these adaptor proteins play a central role in the activation of the map - kinase cascade ( 24 ). as can be seen from the data ( fig2 ), the 66 kda shc exhibited the most prominent association to the autophosphorylated igf - i receptor in response to the insulin molecules . asp b10 insulin and hmr 1153 induced a comparable association of the 66 kda shc to the igf - i receptor that was about 3 - 4 fold higher compared to that induced by insulin and hmr 1964 . most importantly , no significant difference was observed between human insulin and hmr 1964 at this level of the signaling cascade . it should be noted that the much higher autophosphorylation of the igf - i receptor by hmr 1153 does not lead to an appropriately strong interaction with shc ( fig2 ). for all experiments equal loading was ensured by re - probing the blots with an anti - igf - i - receptor - antibody ( not shown in the fig .). to further investigate the interaction between the igf - i receptor and the shc proteins , tyrosine phosphorylation of these intracellular substrates was determined after stimulation of k6 myoblasts with human insulin or the insulin analogs . for this assay the cell extracts were subjected to immunoprecipitation with an anti - shc - antibody and the resulting precipitates were analyzed by immunoblotting with an anti - phosphotyrosine antiserum . as shown in fig3 , the k6 myoblasts express only two shc proteins , the 66 kda and the 52 kda isoform . the two insulin analogs hmr 1153 and asp b10 insulin induced the strongest tyrosine phosphorylation of the two shc isoforms . again , the analog hmr 1964 was comparable to human insulin inducing a much lower shc phosphorylation ( fig3 ). quantification of tyrosine phosphorylation of the most prominent 52 kda shc isoform from multiple experiments demonstrated a 7 fold increase in the level of shc phosporylation after stimulation with hmr 1153 and 5 fold response after treatment with asp b10 insulin . after stimulation with either hmr 1964 or human insulin an approximately 2 fold increase in shc phosphorylation was observed ( fig3 ). the same results were obtained regarding the phosphorylation level of the 66 kda shc isoform ( fig3 ). it has been reported that igf - i receptor internalization regulates signaling via the map - kinase pathway but not the insulin receptor substrate - 1 pathway ( 24 ). taking into account a high rate of internalization of the analogs asp b10 insulin and hmr 1153 and the stronger interaction with the shc proteins , it was anticipated that these two analogs may induce a strong activation of the p42 / 44 map - kinase in the k6 myoblasts . activation of the map - kinases was assessed by monitoring the phosphorylation state of these proteins using phospho specific map - kinase antiserum that detects tyrosine phosphorylated erk1 and erk2 . the data shown in fig4 indicate that k6 cells express both map - kinases , the 44 kda and the 42 kda isoform . the phosphorylation of both isoforms was strongly activated after stimulation of cells with either hmr 1153 or asp b10 insulin . quantification of the data showed a 7 and 5 fold activation for erk1 and erk2 , respectively , after treatment with hmr 1153 . asp b10 insulin was less potent than hmr 1153 but still produced a significantly higher response compared to human insulin and hmr 1964 . the latter analog also produced the lowest response of map - kinase activation that was significantly different from human insulin . the erk½ signaling pathway plays a critical role in the regulation of cellular proliferation and differentiation ( 25 ). cellular proliferation of k6 myoblasts was also determined in response to human insulin and the different analogs by monitoring dna synthesis using the incorporation of bromo - deoxyuridine and a highly sensitive chemiluminescence immunoassay . serum - starved myoblasts responded with a 4 fold increase in brdu - incorporation when stimulated with 10 % fcs or igf - i for 16 h ( fig5 ). both asp b10 insulin and hmr 1153 were equipotent inducing a 2 - 3 fold increase in dna synthesis but were significantly less potent that igf - i . the smallest response ( about 50 %) was observed for both human insulin and hmr 1964 ( fig5 ). these two molecules were significantly less potent than asp b10 insulin and hmr 1153 . thus , the growth promoting activity of the insulin analog hmr 1964 is identical to that of human insulin and is completely consistent with the insulin - like activation of the shc / map - kinase cascade by this analog . the data obtained demonstrates that the analogs hmr 1153 and asp b10 insulin strongly activate the map - kinase pathway via a prominent stimulation of shc phosphorylation . in contrast , human insulin and the analog hmr 1964 exerted a much weaker effect on this pathway . to further dissect the signaling properties of the different insulin analogs , their effects on the tyrosine phosphorylation of irs - ½ in k6 myoblasts was determined . the cells were stimulated with insulin or insulin analogs for 10 min as described before , and irs - 1 or irs - 2 were immunoprecipitated and immunoblotted with anti - phosphotyrosine antibodies . as shown in fig6 , human insulin produced the strongest tyrosine phosphorylation of both irs - 1 and irs - 2 , whereas the analogs asp b10 insulin and hmr 1153 induced a less pronounced stimulation of both irs - proteins . remarkably , the analog hmr 1964 produced only a marginal phosphorylation of irs - 1 , but produced a very strong phosphorylation of irs - 2 that was similar to that seen after stimulation with human insulin ( fig6 ). quantification of the western blots ( fig6 — right panel ) demonstrated an approximately 30 fold and a nearly 20 fold response after treatment with human insulin for irs - 1 and irs - 2 , respectively . the analog hmr 1964 exerted a marginal 2 fold effect on the activation of irs - 1 , but induced a 20 fold increase of the irs - 2 phosphorylation being as effective as human insulin ( fig6 ). since the preferred stimulation of irs - 2 by a modified insulin molecule was unexpected , these experiments were repeated in a different cell system . proliferating primary human skeletal muscle cells were stimulated with human insulin and the different analogs using exactly the same protocol as outlined above for the k6 myoblasts , and the tyrosine phosphorylation of irs - 1 and irs - 2 was analyzed by immunoblotting ( fig7 ). as seen before , human insulin produced a very strong phosphorylation of both irs - 1 and irs - 2 with the analog asp b10 insulin being equipotent to the regular insulin molecule . however , again the insulin analog hmr 1964 produced a marginal phosphorylation of irs - 1 and a very strong tyrosine phosphorylation of irs - 2 that was even significantly higher than that seen after stimulation of cells with human insulin ( fig7 ). thus , the preferential activation of irs - 2 by the insulin analog hmr 1964 represents a unique property of this molecule that is also effective in human cells . it may be argued that the special effect of hmr 1964 is mediated by the igf - i receptor and thus could be limited to myoblastic cells . the tyrosine phosphorylation of irs - 1 and irs - 2 in response to the different insulin analogs in primary adult cardiomyocytes was studied . these cells express a high level of insulin receptors but much less igf - i receptors and have been extensively used for studies on insulin signaling and insulin action ( 19 , 26 , 27 ). all experiments were conducted under the same conditions as described before for k6 cells and human myoblasts . as shown in fig8 , also in this cell system human insulin produced a strong phosphorylation of both irs - 1 and irs - 2 , whereas asp b10 insulin and hmr 1153 were less effective . again , tyrosine phosphorylation of irs - 1 was only marginally activated by hmr 1964 ( 2 fold ). however , this analog produced an 18 fold increase of the tyrosine phosphorylation of irs - 2 reaching the same level as that seen with human insulin ( fig8 ). these data confirm that the novel analog hmr 1964 preferentially signals along the irs - 2 pathway , both in myoblasts and differentiated muscle cells . the lack of irs - 1 activation by hmr 1964 may limit the metabolic activity of this analog . to address this issue , the stimulation of 3 - o - methylglucose transport by this analog in direct comparison to human insulin using the adult cardiomyocyte system was measured . as presented in fig9 , the initial rate of glucose transport was increased 3 and 4 . 4 fold in response to 5 and 500 nm insulin , respectively . essentially the same response was observed after treatment of cells with hmr 1964 ( fig9 ). thus , activation of irs - 2 appears to be sufficient for propagating downstream signaling to glucose transporters in muscle cells . effect of insulin , insulin analogs and igf - i on cytokine - induced apoptosis in pancreatic β - cells the clonal glucose sensitive rat insulinoma cell line ins - 1 is described by m . asfari et al ., endocrinology 130 ( 1992 ) 167 - 178 . bovine serum albumin ( bsa , fraction v , fatty acid free ) was obtained from boehringer mannheim ( mannheim , germany ). fetal calf serum ( fcs ), dulbecco &# 39 ; s modified eeagle medium ( dmem ), rpmi 1640 medium , non - essential amino acids and penicillin / streptomycin were provided by gibco ( eggenstein , germany ). the cell death detection elisa kit was obtained from roche diagnostics ( mannheim , germany ). all other chemicals were of the highest grade commercially available . the rat insulinoma cell line ins - 1 was grown in rpmi 1640 medium supplemented with 10 % heat - inactivated fetal calf serum ( fcs ), 500 u / ml penicillin , 50 μg / ml streptomycin , 2 mmol / l glutamine and 50 μmol / l 2 - mercaptoethanol and passaged by trypsinization . functional integrity of the cells was confirmed by measuring glucose - stimulated insulin secretion . ins - 1 cells were routinely seeded at 3 . 5 × 10 5 cells / well of a 12 - well plate for cell death detection experiments and used on day 4 at 60 - 70 % confluence . subconfluent ins - 1 cells were incubated for 2 h in rpmi 1640 medium ( eggenstein , germany ) without fcs supplemented with 0 . 5 % bsa . after this serum free period the medium was removed and replaced with fresh rpmi 1640 containing 5 % fcs and 0 . 5 % bsa . cells were then incubated with the cytokine combination interleukin - 1β ( il - 1β ) and interferon - γ ( ifn - γ ) in the absence or presence of the different insulin peptides ( 500 nm ) or igf - i ( 10 nm ) for 24 h at 37 ° c . after removing this medium , the cells were washed twice with ice cold pbs and lysed . the induced apoptosis was measured by the specific determination of mono - and oligonucleosomes in the cytoplasmatic fraction of cell lysates using a cell death detection elisa kit ( roche ). the assay is based on a quantitative sandwich - enzyme - immunoassay - principle using mouse monoclonal anti - histone and anti - dna peroxidase antibodies . the relative degree of apoptotic cell death was photometrically determined by measuring the peroxidase activity of the immunocomplexes at 405 nm . in the following table , results are expressed as % inhibition of cytokine - induced apoptosis as measured by nucleosomal release . data are mean values ± sem of 4 - 5 separate experiments . it was observed that the novel rapid acting analog lys b3 , glu b29 insulin ( hmr 1964 ) was able to generate a preferential activation of the irs - 2 signaling pathway in muscle cells , concomitantly exhibiting the same mitogenic and metabolic properties as regular human insulin . this is the first report on the selective or preferential activation of irs - 2 by an insulin analog . modification of the b26 - b30 region of the insulin molecule has been extensively used to produce insulin analogs with reduced self - association being suitable as rapid acting insulin molecules ( 3 - 5 ). however , modifications within this domain of the insulin molecule are known to increase the affinity of a given analog for the igf - i receptor , finally leading to an enhanced mitogenic activity and a potential safety risk of these compounds related to long - term use ( 9 , 12 , 15 , 28 ). this concept has been reassessed by performing a detailed analysis of the signaling and mitogenic properties of the two analogs lys b3 , glu b29 insulin and lys b3 , ile b28 insulin ( hmr 1153 ) in rat and human myoblasts expressing a high level of igf - i receptors . the data suggest that in addition to the binding affinity and the occupancy time at the receptor ( 14 ), initial steps of receptor activation and / or processing may also contribute to trigger specific downstream signaling pathways by the insulin molecule . both irs - 1 and shc have been implicated in the activation of the map kinase pathway by igf - i and insulin ( 29 , 30 ), a signalling event with central importance for the control of cellular growth and differentiation by these hormones ( 31 ). more recently , a differential interaction of the ptb - domains of shc and irs - 1 with the igf - i receptor has been reported ( 32 ), and a sustained tyrosine phosphorylation of shc in response to igf - i was found to correlate with enhanced map kinase activation and growth of human neuroblastoma cells ( 33 ), but was unrelated to the tyrosine phosphorylation of irs - 2 . as shown by chow et al . ( 24 ), igf - i receptor internalization is required for signaling via the shc / map kinase pathway , but not the irs - 1 pathway . consistently , our data show a good correlation between the internalization of the insulin analogs and the activation of the map kinase pathway in the k6 myoblasts . thus , hmr 1153 exhibited a 3 - 4 fold higher internalization when compared to hmr 1964 , and this resulted in a 3 - 4 fold higher shc phosphorylation and erk½ activation in response to hmr 1153 . furthermore , the low rate of internalization of hmr 1964 correlates with a marginal activation of erk½ by this analog that is even lower than that induced by insulin . it has also been demonstrated that sustained receptor binding decreases endosomal insulin degradation , resulting in enhanced signaling from this intracellular compartment ( 34 ). this would explain the strong activation of the map kinase by asp b10 insulin , since this analog exhibits a moderate internalization combined with a very low degradation ( see table 2 ). human insulin and hmr 1964 produced a moderate activation of the shc / map kinase pathway and of dna synthesis in the k6 cells . also note that under the same conditions insulin induces a prominent activation of both irs - 1 and irs - 2 . thus , the data support the notion that tyrosine phosphorylation of shc , most likely leading to formation of the shc - grb2 complex , represents a key step in igf - i receptor signaling to the map kinase pathway ( 30 , 33 ). it is also evident that the prominent activation of irs - 2 by hmr 1964 does not mediate map kinase activation . this is consistent with the view that irs - 2 is of major importance for mediating metabolic events ( 35 - 37 ). interestingly , the autophosphorylation of the igf - i receptor in response to hmr 1964 was comparable to that seen after stimulation of cells with asp b10 insulin ; however , association of shc with the igf - i receptor was much stronger after stimulation with asp b10 insulin . furthermore , hmr 1153 produced a very strong autophosphorylation of the igf - i receptor but the same association with shc when compared to asp b10 insulin . these observations demonstrate that the insulin / receptor interaction may control the specificity of downstream signaling by a combination of several mechanisms . in addition to binding , this may include : i ) the internalization and processing of the ligand receptor complex , ii ) the half - life of the receptor complex , and iii ) a differential phosphorylation pattern of the igf - i / insulin receptor in response to the different analogs . the latter possibility fits with the observation that shc and irs - 1 employ functionally distinct mechanisms to recognize tyrosine phosphorylated receptors ( 32 , 38 ). the mechanism by which ligands interact with their receptors to mediate signaling is still not understood in great detail at the molecular level . the ligand - induced receptor activation has been suggested to involve a conformational switch in the quaternary structure upon ligand binding with movements of the extracellular alpha parts and a congregation of the cytoplasmic tyrosine kinase regions to enable activation ( 39 , 40 ). thus it may be speculated that differences in the structural changes of the receptor induced by the analogs possibly affect the receptor phosphorylation pattern leading to a differential interaction with downstream substrates and thus resulting in divergent action profiles . different interactions between the receptor and the analogs could possibly switch the receptor conformation to a state that allows the formation of a stable complex between the receptor and a specific substrate . a hypothetical explanation for the discrepancies in binding potency and receptor phosphorylation obtained by the analogs is that they bind to the receptor in a different manner locking the subunits of the receptors in distinct conformation states and thus affecting its phosphorylation pattern . however , extensive and detailed functional and high resolution structural studies will be necessary to confirm this hypothesis . human insulin exerted the most prominent activation of irs - 1 and irs - 2 in both rat and human myoblasts and adult cardiomyocytes . hmr 1153 and asp b10 insulin were much less effective at this level of the insulin signaling cascade despite a 2fold higher proliferative activity of these analogs in the k6 myoblasts . thus shc / map kinase signaling is the major determinant of the mitogenic activity of insulin analogs . surprisingly , the analog hmr 1964 produced only a marginal activation of irs - 1 in the three cell systems , but a prominent activation of irs - 2 , that was even significantly different from regular insulin in the human myoblasts . this may be a specific property of myoblastic cells expressing a high level of the igf - i receptor . however , as shown here , hmr 1964 produces an exclusive activation of irs - 2 in adult rat cardiomyocytes , a cell expressing a high level of insulin receptors ( 26 ). furhtermore , the activation of irs - 2 is sufficient to produce a full metabolic response in the adult cardiomyocyte , since hmr 1964 was equipotent to insulin in activating glucose transport in these cells . consistently , irs - 1 but not irs - 2 has been found to induce the ras - map kinase signaling required for fetal brown adipocyte proliferation ( 37 ). further , irs - 1 represents the main substrate mediating the mitogenic actions of igf - i receptors in hepatocytes ( 36 ), whereas irs - 2 is a dominant regulator of the metabolic effects of insulin in l6 muscle cells ( 35 ). the molecular basis of the preferred activation of irs - 2 by hmr 1964 is presently unknown . an alignment of the predicted amino acid sequence of murine irs - 1 and irs - 2 revealed two conserved regions , the ih1 ( ph ) and the ih2 ( ptb ) domains ( 41 ). a third region found in irs - 1 , called sain domain , is poorly conserved between irs - 1 and irs - 2 ( 42 ). recent studies have identified a novel domain of strong interaction in the central region of irs - 2 , localized between amino acids 591 and 786 , which is absent in irs - 1 . this irs - 2 specific region was found to be independent of the npx ( p ) y - motif . however it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop ( tyr1146 , tyr1150 and tyr1151 ). importantly this novel domain may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances irs - 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