| Mass,strength and function of skeletal muscle will decline with the increase of age,while vivo and vitro experiments have indicated that aging can lead to the decrease of glucose transport in skeletal muscle and increase insulin resistance.Insulin resistance is associated with many prevalent age-related diseases,including type 2 diabetes,hypertension,cardiovascular disease,some cancers.Skeletal muscles make up 40% of the body's mass in mammals including humans and rodents and accounts for the largest amount of insulin-stimulated blood glucose clearance.In particular,skeletal muscle can dispose of as much as 85% of insulin-induced clearance of the blood glucose load.Defects in skeletal muscle insulin signaling which are responsible for causes of insulin resistance.Skeletal muscle from old individuals remains responsive to improved insulin sensitivity with various interventions,advancing the understanding of these interventions may offer useful insights for improving health at older ages.Either calorie restriction(CR;consuming 60-65% of ad libitum,AL,intake)or acute exercise can independently improve insulin sensitivity in adult rats.Current knowledge about the independent effects of CR or acute exercise on the role of insulin signaling in the regulation of muscle glucose uptake during old age is limited,and apparently nothing has been published about the combined effects of CR and acute exercise on insulin signaling in muscles,regardless of age.Obejective: Skeletal muscle with different fiber types were used as entry point in this study,the current study was designed to provide new insights into the mechanisms for both the independent and combined benefits of CR and acute exercise on insulin signaling and glucose uptake by insulin-stimulated muscle in fast-twitch and slowtwitch in old age.The specific aims were: 1)to determine the independent and combined benefits of CR and acute exercise on glucose uptake in fast-twitch and slowtwitch muscle of old rats.2)to elucidate potential mechanisms for CR and/or acute exercise effects on insulin-stimulated glucose uptake which will focus on insulin signaling in fast-twitch and slow-twitch muscle.The second aim of the current study were: 1)to evaluate the benefits of CR for insulin-stimulated glucose uptake with CR initiated in older rats;2)to gain insights into the mechanisms for increased insulinstimulated glucose uptake when CR is initiated in old age and evaluate the role of Akt phosphorylation and Akt2 activity in the CR-induced enhancement of glucose uptake in muscles from old rats and reveal some Akt's substrats that are potential involved in glucose uptake in muscle.Methods: 1.Mechanisms for both the independent and combined benefits of CR and acute exercise on glucose uptake in fast-twitch and slow-twitch muscle of old rats.(Study I and Study II)The study was subjected 30 month-old Fischer 344 x Brown Norway(FBN)male rats(CR is initiated at 3 month-old),rats were intervened with CR and/or acute exercise.Animals were randomly assigned into four groups: sedentary and AL(AL-SED,n=8),sedentary and CR(CR-SED,n=8),3 hours post-exercise and AL(AL-3h PEX,n=10),3 hours post-exercise and CR(CR-3h PEX,n=11).CR is initiated at 3 month-old until 30 month-old.The animals were fasted for at least 12 hours before the experiment.Exercised rats swam in a barrel filled with water of 35 oC.The exercise protocol consisted of nine bouts of swimming(10 minute duration per bout)with 10 minute rest intervals separating each exercise bout.After 90 minutes of total exercise,exercising rats were dried and returned to their cages without food,epitrochlearis and soleus muscles were dissected from anesthetized time-matched sedentary and exercised rats at 3-4 hours after the exercising group had completed the protocol.Epitrochlearis and soleus muscles were incubated with either no insulin(basal)or a submaximally effective concentration of insulin buffer.After incubation,there are 8 groups for the muscles: AL-SED-Basal,AL-SED-Insulin,CR-SED-Basal,CR-SED-Insulin,AL-3h PEX-Basal,AL-3h PEX-Insulin,CR-3h PEX-Basal,CR-3h PEX-Insulin,n?8 for each group.Muscles were freeze-clamped using aluminum tongs cooled in liquid nitrogen,and stored at-80 oC for later processing and analysis.1)The effect of CR on the body weight of rats;2)2-DG radiolabeled with 3H and 14 C was used to look at rat of glucose transport in the muscles;3)Radiolabeled with 32 P was used for IRS-1/PI3 K activity;4)western blot was used for analyzing abundance and activation of key insulin signaling protein that regulate glucose uptake: IR,Akt,AS160,TBC1D1 and phosphorylation of IR Try1146,Akt Ser473,Akt Thr308,AS160 Ser588,AS160 Thr642;abundance of AMPK,ACC,FLNc and phosphorylation of AMPK Thr172,ACC Ser79,FLNc Ser2213;abundance of GLUT4,GLUT1 and hexokinase II(proteins that control glucose transport and phosphorylation).5)Coimmunoprecipitation was use to evaluate Akt associated with HSP90,PP2 A,APPL1.2.The role of Akt phosphorylation and Akt2 activity in the CR-induced enhancement of glucose uptake in muscles from old rats(Study III)40 male 23 month-old FBN rats were randomly assigned into four groups: ad libitum(AL)and calorie restriction(CR)with 8 rats for each group.The CR group was restricted to 65% of AL daily intake for 8 weeks..After the 8 weeks CR period,the AL and CR rats(aged ~25 months-old)were deeply euthanized by an intraperitoneal injection of sodium pentobarbital,and then epitrochlearis muscles were dissected out.Mucles were incubated without(Basal)or with a submaximally effective insulin(insulin)or Akt inhibitor(MK-2206).After incubation,there are 5 groups for the muscles: AL-Basal,AL-Insulin,CR-Basal,CR-Insulin,CR-Insulin+MK2206,n=8 for each group.Muscles were freeze-clamped using aluminum tongs cooled in liquid nitrogen,and stored at-80 oC for later processing and analysis.1)The effect of CR on the body weight of rats;2)2-DG radiolabeled with 3H and 14 C was used to look at rat of glucose transport in the muscles;3)Radiolabeled with 32 P was used for Akt2 activity activity;4)immunoprecipitation was use to evaluate Akt2 Ser474 and Thr309 phosphorylation;5)western blot was used for analyzing total Akt, Akt2 and AMPK protein abundance,total Akt Ser473 and Thr308,AMPK Thr172 phosphorylation;abundance and site-selective phosphorylation of four Akt substrates: AS160 on Ser588 and Thr642,TBC1D1 on Thr596,PRAS40 on Thr244 and FLNc on Ser2213.Results: 1.The study was subjected 30 month-old Fischer 344 x Brown Norway(FBN)male rats(CR is initiated at 3 month-old),rats were intervened with CR and/or acute exercise.1)The body weight of CR group was decreased compared AL groups.2)CR alone versus AL sedentary controls induced greater insulin-stimulated glucose uptake in both fast-twitch and slow-twitch muscle of old rats(P<0.001);In fast-twitch muscles,exercise alone versus AL sedentary controls induced greater insulinstimulated glucose uptake(P<0.05)and combined CR and exercise versus either treatment alone caused an additional increase in insulin-stimulated glucose uptake(P<0.001).Exercise alone versus AL sedentary and Combined CR and exercise did not further increase insulin-stimulated glucose uptake in slow-twitch muscle of old rats.3)There was no effect of independent and combined benefits of CR and acute exercise on the phosphorylation of IR and IRS-PI3 K activity in both fast-twitch and slow-twitch muscle of old rats.4)In fast-twitch muscles,CR alone increased insulin-stimulated phosphorylation of Akt Ser473(P<0.01);Akt Thr308(P<0.001),AS160 Ser588(P<0.05),AS160 Thr642(P<0.05),FLNc Ser2213(P<0.05),AMPK Thr172(P<0.01);there was no significant effect of CR on the phosphorylation of TBC1D1Thr596,ACC Ser79.In slow-twitch muscles,CR alone increased insulin-stimulated phosphorylation of Akt Ser473(P<0.05),Akt Thr308(P<0.05),AS160 Ser588(P<0.01),TBC1D1 Thr596(P<0.001),but not phosphorylation of AS160 Thr642,FLNcSer2213,AMPK Thr172,ACC Ser79.5)In fast-twitch muscles,exercise alone results greater insulin-stimulated phosphorylation of Akt Ser473(P<0.05),Akt Thr308(P<0.05),FLNc Ser2213(P<0.05). However,the phosphorylation of AS160 Ser588,AS160 Thr642,TBC1D1Thr596,ACC Ser79,AMPK Thr172 was not increased after acute exercise.In slow-twitch muscles,exercise alone did not increase insulin-stimulated phosphorylation of Akt Ser473,Akt Thr308,AS160 Ser588,AS160 Thr642,TBC1D1 Thr596,ACC Ser79,AMPK Thr172,FLNcSer2213.6)In fast-twitch muscles,CR and acute exercise further elevated the insulin-stimulated phosphorylation of Akt Ser473(P<0.05),Akt Thr308(P<0.001),FLNcSer2213(P<0.05).but not phosphorylation of AS160 Ser588,AS160 Thr642,TBC1D1 Thr596,ACC Ser79,AMPK Thr172.In slow-twitch muscles,the combined CR and exercise group was characterized by elevated p AMPK Thr172(P<0.05)and was accompanied by no significant combined CR and exercise effect on the phosphorylation of any of Akt Ser473,Akt Thr308,AS160 Ser588,AS160 Thr642,TBC1D1 Thr596,ACC Ser79 ?FLNcSer2213.7)In fast-twitch muscles,CR alone increased Akt associated with HSP90(P<0.05)and exercise alone decreased Akt associated with PP2A(P<0.05).However,combined CR and acute exercise did not further increase Akt associated with HSP90 and APPL1.In slow-twitch muscles,exercise alone,CR alone and combined CR and acute exercise did not further increase Akt associated with PP2 A,HSP90 and APPL1.8)In fast-twitch muscles,HKII protein abundance was decreased by CR alone(P<0.01).There was a modest,but significant increase in GLUT4 protein abundance after acute exercise(P<0.01).In slow-twitch muscles,there was no CR alone and exercise alone effect on GLUT4 and HKII protein abundance.2.The study were subjected from 23 month old FBN rats either fed AL or CR in old age,isolated muscles were incubated with MK-2206.The results: CR groups versus AL groups resulted in: 1)body weight was significant decreased(P<0.05);2)greater insulin-stimulated glucose uptake(P<0.05);3)that was accompanied by significantly increased insulin-mediated activation of Akt2 as indicated by greater phosphorylation on both Thr309 and Ser474(P<0.001)along with greater Akt2 activity(P<0.01);4)enhanced phosphorylation of Akt on both Thr308 and Ser473(P<0.001);5)concomitant with enhanced phosphorylation of several Akt substrates,including Akt Substrate of 160 k Da on Thr642(P<0.01),filamin C on Ser2213(P<0.01)and proline-rich Akt substrate of 40 k Da on Thr246(P<0.001),but not TBC1D1 on Thr596;and 6)each of the CR-effects was eliminated by MK-2206.Conclusion: 1.The consistent effect of CR on insulin-stimulated glucose in the fast-twitch muscles(epitrochlearis)and fast-twitch muscles(soleus)from older rats corresponds with the results of many studies with younger rodents that have found that CR can improve insulin-stimulated glucose uptake in muscles with differing fiber type profiles.Taken together,these studies have revealed CR's very robust effect on insulin-stimulated glucose uptake in isolated muscles of diverse fiber type compositions across a wide range of the adult lifespan.2.The mechanism for enhanced insulin-stimulated glucose uptake in fast-twitch and slow-twitch muscles is overlapping and also different.CR alone increase Akt phosphorylation on two sits Thr308 and Ser473 in both fast-twitch and slow-twitch muscles.But in the fast-twitch muscles,AS160 serine and threonine phosphorylation was increased by Akt activation,and in the slow-twitch muscles,TBC1D1 phosphorylation was increased by Akt.The influence of CR on Akt phosphorylation was not accounted fo by greater change in Akt banding to HSP90,PP2 A and APPL1.3.The effect of exercise alone on the insulin-stimulated glucose uptake in fast-twitch and slow-twitch muscle is different.In fast-twitch muscles,exercise alone versus AL sedentary controls induced greater insulin-stimulated glucose uptake.Increased phosphorylation of Akt Thr308 and Ser473,FLNc and decreased Akt associated with PP2 A are possible involved in the mechanism.However,exercise alone versus AL sedentary did not further increase insulin-stimulated glucose uptake and activation of Akt,AS160,TBC1D1,FLnc in slow-twitch muscle of old rats.Taken together,it is possible that different fiber type composition and recruited fiber account for the different insulin-stimulated glucose uptake.4.There was significant difference of combined effects of calorie restriction and acute exercise on Insulin-stimulated glucose uptake in fast-twitch and slow-twitch muscles.In fast-twitch muscles,combined CR and exercise versus either treatment alone caused an additional increase in insulin-stimulated glucose uptake.Other than Akt phosphorylation,the only signaling outcomes found to be greater for muscles from the combined CR and exercise group versus either the CR or exercise alone groups were the phosphorylation of AMPK Thr172 and FLNc Ser2213.No further increase in AS160 phosphorylation was found on either phospho-site when CR was combined with exercise.It was suggested that the further increase in glucose uptake in the combined group was secondary to other changes(e.g.,GLUT4 vesicle trafficking).In slow-twitch muscles,combined CR and exercise did not further increase insulin-stimulated glucose uptake and no further increase of Akt,AS160,TBC1D1,FLNc phosphorylation was found in slow-twitch muscle of old rats.These results implicated it is possible that differences in metabolic properties for muscles with differing fiber type composition account for the different insulin-stimulated glucose uptake.5.Analysis of the results for the independent and combined interventions(CR,exercise,or CR combined with exercise)revealed a close correspondence between the increases in glucose uptake and Akt phosphorylation.These results provide evidence that the improved insulin-stimulated glucose uptake may be secondary,at least in part,to greater activation of Akt,presumably as a consequence of greater phosphorylation of one or more Akt substrate.Greater AS160 phosphorylation was evident with CR alone,but the results for exercise alone or combined CR and exercise revealed the need to consider other Akt substrates.In this context,it was interesting that greater FLNc phosphorylation tracked with the levels of both Akt phosphorylation and insulinstimulated glucose uptake in all treatment groups.6.There is significant effect on the insulin-stimulated muscles even if CR is initial in old age rats.Attaining greater Akt2 and Akt activity and AS160 phosphorylation would be expected to favor greater insulin-stimulated glucose uptake,but enhanced phosphorylation of other Akt substrates FLNc and PRAS40 may also contribute to elevated insulin sensitivity.MK-2206 eliminated the CR effect on Akt2 phosphorylation and activity,and the significant effects of CR on phosphorylation of AS160,FLNc and PRAS40 were also eliminated concomitant with elimination of CR's effect on insulin-stimulated glucose uptake.These observation suggested Akt and Akt2 was demonstrated to be essential for CR's full effect on insulin-stimulated glucose uptake by muscle. |
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