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Am J Physiol Endocrinol Metab 293: E1782-E1788, 2007. First published October 9, 2007; doi:10.1152/ajpendo.00531.2007
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In vitro simulation of calorie restriction-induced decline in glucose and insulin leads to increased insulin-stimulated glucose transport in rat skeletal muscle

Edward B. Arias and Gregory D. Cartee

Muscle Biology Laboratory, Division of Kinesiology, University of Michigan, Ann Arbor, Michigan

Submitted 15 August 2007 ; accepted in final form 4 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In vivo calorie restriction [CR; consuming 60% of ad libitum (AL) intake] induces elevated insulin-stimulated glucose transport (GT) in skeletal muscle. The mechanisms triggering this adaptation are unknown. The aim of this study was to determine whether physiological reductions in extracellular glucose and/or insulin, similar to those found with in vivo CR, were sufficient to elevate GT in isolated muscles. Epitrochlearis muscles dissected from rats were incubated for 24 h in media with glucose (8 mM) and insulin (80 µU/ml) at levels similar to plasma values of AL-fed rats and compared with muscles incubated with glucose (5.5 mM) and/or insulin (20 µU/ml) at levels similar to plasma values of CR rats. Muscles incubated with CR levels of glucose and insulin for 24 h had a subsequently greater (P < 0.005) GT with 80 µU/ml insulin and 8 mM [3H]-3-O-methylglucose but unchanged GT without insulin. Reducing only glucose or insulin for 24 h or both glucose and insulin for 6 h did not induce altered GT. Increased GT after 24-h incubation with CR levels of glucose and insulin was not attributable to increased insulin receptor tyrosine phosphorylation, Akt serine phosphorylation, or Akt substrate of 160 kDa phosphorylation. Nor did 24-h incubation with CR levels of glucose and insulin alter the abundance of insulin receptor, insulin receptor substrate-1, GLUT1, or GLUT4 proteins. These results provide the proof of principle that reductions in extracellular glucose and insulin, similar to in vivo CR, are sufficient to induce an increase in insulin-stimulated glucose transport comparable to the increase found with in vivo CR.

diet restriction; insulin signaling; GLUT4; Akt; Akt substrate of 160 kDa

ALTERED SKELETAL MUSCLE GLUCOSE transport can contribute to important systemic changes in glucose homeostasis. For example, skeletal muscle insulin resistance for glucose uptake is an early and essential defect for the subsequent development of the metabolic syndrome and type 2 diabetes mellitus (19, 20). Previous studies have demonstrated that prior exposure of skeletal muscle to large changes in extracellular glucose and insulin concentrations over a period of hours can lead to altered glucose transport. However, only a few studies have characterized the effects of moderate physiological changes in glucose on glucose transport. In cultured myocytes, increasing glucose from ~5 to ~8–10 mM for 24 h did not markedly reduce glucose transport (31, 38). In isolated rat soleus muscles, there was also little difference in glucose uptake without insulin after 3 h of incubation with ~5 compared with ~8 mM glucose (38), but it is possible that a more prolonged incubation would reveal an effect in this range of glucose concentrations.

Knowledge is also limited about prolonged exposure to different physiological insulin levels. When rat epitrochlearis muscles were incubated for 5 h with a physiological insulin dose and a range of glucose levels (1–36 mM), there was a glucose concentration-dependent decline in glucose transport (41). Muscles exposed to differing glucose concentrations (0, 5, or 25 mM) with physiological insulin during a 2-h hindlimb perfusion had less of a reduction in subsequent glucose transport than muscles exposed to the same glucose with supraphysiological insulin (26). At least for moderate vs. very high insulin, prolonged exposure to higher insulin exacerbates glucose-induced decrements in glucose uptake.

Hyperglycemia and hyperinsulinemia can clearly lead to impaired glucose uptake, but it is also valuable to understand the effects of reducing glucose and/or insulin moderately below usual levels. Calorie restriction (CR; consuming ~60% of ad libitum intake), which results in small reductions in plasma glucose (~10–25%) and somewhat greater decrements in plasma insulin (~40–80%) (29, 30), can improve insulin sensitivity in many species, including humans (1) and rats (25). The enhanced in vivo insulin action is attributable, in part, to elevated insulin-stimulated glucose transport with unaltered basal glucose transport in skeletal muscle (11, 12, 15, 16, 23, 32–34).

The major goal of this study was to determine whether changes in glucose and/or insulin similar to those found with CR are sufficient to induce an increase in insulin-stimulated glucose transport of skeletal muscle. Epitrochlearis muscles from rats underwent a prolonged (up to 24 h) exposure to physiologically relevant concentrations of glucose (5.5 or 8 mM) and insulin (20 or 80 µU/ml) before the measurement of glucose transport. We hypothesized that exposing muscles to reductions in only insulin or only glucose would not alter subsequent glucose transport. We also hypothesized that exposing muscles to reductions in both insulin and glucose would result in subsequently increased insulin-stimulated, but not basal, glucose transport. The in vivo CR-induced increase in insulin-stimulated glucose transport appears to be partly attributable to increased Akt phosphorylation (32–34) despite unaltered GLUT4 glucose transporter abundance (12, 22). Therefore, we further hypothesized that the enhanced insulin action in muscles that were incubated with reduced glucose and insulin would be accompanied by elevated insulin-stimulated Akt phosphorylation but not increased GLUT4 abundance.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Unless otherwise noted, all chemicals were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Hanover Park, IL). Human recombinant insulin was obtained from Eli Lilly (Indianapolis, IN). Reagents and apparatus for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Richmond, CA). Protein concentration was measured using the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL). Enhanced chemiluminescence (ECL; West Dura Extended Duration Substrate, Pierce, no. 34075) was used to visualize protein bands. Anti-phospho-Ser473 Akt (no. 9271), anti-GLUT4, and anti-phospho-(Ser/Thr) Akt substrate (anti-PAS; no. 9611) were purchased from Cell Signaling Technology (Danvers, MA). Anti-GLUT1 (no. CBL242) and anti-insulin receptor substrate-1 (anti-IRS1; no. 06-248) were purchased from Upstate Biotechnology (Charlottesville, VA). 3-O-methyl-[3H]glucose ([3H]3-MG) was from New England Nuclear Life Science Products (Boston, MA), and [14C]mannitol was from Amersham Pharmacia Biotechnology (Piscataway, NJ). Dulbecco's modified Eagle's medium (DMEM; no. 31600), gentamycin, streptomycin, and penicillin were purchased from Invitrogen (Carlsbad, CA). Total insulin receptor (IR) abundance (no. KHR9111) and IR tyrosine phosphorylation [pYpY1162/1163] (no. KHR9131) ELISA kits were purchased from BioSource International (Camarillo, CA). Teflon air filters (no. DDF02T1750) were purchased from GE Osmonics (Minnetonka, MN). Culture media filters were purchased from Nalge Nunc International (Rochester, NY).

Animal treatment. Procedures for animal care were approved by the University of Michigan Committee on Use and Care of Animals. Female Fischer 344 rats (7–8 wk old, ~170 g; Harlan, Indianapolis, IN) were provided with Purina rat chow (no. 5001) and water ad libitum. On the experimental day between 1200 and 1400, the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). On loss of pedal reflexes, epitrochlearis muscles were quickly excised and transferred into vials containing the appropriate incubation media.

In vitro muscle incubations. The incubation procedures summarized in Fig. 1 are modifications of those previously used with rat epitrochlearis muscles incubated for up to 9 h (24) or 19 h (5). Following dissection, muscles were incubated for 1, 6, or 24 h (incubation step 1) in flasks containing 2 ml of DMEM supplemented with 2.54 mM CaCl2, 25 mM NaHCO3, 0.1% bovine serum albumin (BSA), glucose (5.5 or 8 mM), insulin (20 or 80 µU/ml), and antibiotics (100 µU/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamycin). All flasks and water used during muscle incubation steps were autoclaved. Previous results indicated that it is important that media include branched chain amino acids (in DMEM) and a physiological insulin concentration when rat epitrochlearis muscles undergo prolonged in vitro incubations to avoid a large increase in basal glucose transport (24). Flasks were placed in a shaking water bath at 35°C and continuously gassed with 95% O2-5% CO2 throughout the experiment. Teflon air filters (Osmonics) were used to minimize aerosol contaminants.


Figure 1
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  		Fig. 1. Schematic diagram of muscle incubation protocol. 3-MG, 3-O-methylglucose; sALgi, simulated (s) ad libitum (AL) values of glucose (g) and insulin (i); sCRgi, simulated (s) calorie reduction (CR) values of glucose (g) and insulin (i); KHB, Krebs-Henseleit buffer.

 
Five different incubation protocols were studied. Each protocol included four incubation steps, and the only procedural differences among the protocols occurred during the first incubation step (Fig. 1). Paired epitrochlearis muscles were dissected from each rat. Within each protocol, comparisons were made between paired muscles that were treated identically except for a single aspect of the incubation conditions during step 1 (i.e., duration of incubation, glucose and/or insulin concentration). The purpose of Protocol 1 was to determine whether the glucose transport rate in a muscle after a relatively brief initial incubation step (1 h) would be maintained by its contralateral muscle after a prolonged initial incubation step (24 h). All muscles in this protocol were incubated with 8 mM glucose and 80 µU/ml insulin during incubation step 1. These glucose and insulin concentrations are within the range of values found in the plasma of ad libitum-fed rats (29, 30, 35, 36, 39). The groups were designated as simulated ad libitum values of glucose and insulin for 1 h (sALgi-1h) or 24 h (sALgi-24h).

For all muscles studied in Protocol 2, the duration of the initial incubation step was 24 h. The purpose of this protocol was to determine the effect of altering only the concentration of glucose during step 1 on subsequent glucose transport rate. One of the paired muscles was incubated as described above for the sALgi-24h treatment. The contralateral muscle was incubated identically, except that during incubation step 1, the glucose concentration was 5.5 mM. This glucose concentration is within the range of plasma values found in rats that are undergoing moderate CR, i.e., that have been consuming ~60% of ad libitum food intake (29, 30, 35, 36, 39). The group with a lower glucose concentration during step 1 was designated as simulated CR values of glucose for 24 h (sCRg-24h).

Protocol 3 compared the effects of altering only insulin during incubation step 1 on subsequent glucose transport. One of the paired muscles was incubated as described above for the sALgi-24h treatment. The contralateral muscle was incubated identically, except the insulin concentration during incubation step 1 was 20 µU/ml. This insulin concentration is within the range of plasma values found in CR rats (29, 30, 35, 36, 39). The group with a lower insulin concentration during step 1 was designated as simulated CR values of insulin for 24 h (sCRi-24h).

Protocol 4 determined the effects of reducing both insulin and glucose concentrations during incubation step 1 on subsequent glucose transport. One of the paired muscles was incubated as described above for the sALgi-24h treatment. The contralateral muscle was incubated identically, except for lower concentrations of glucose (5.5 mM glucose) and insulin (20 µU/ml) during the initial incubation step. The group with lower glucose and insulin concentrations was designated as simulated CR values for glucose and insulin for 24 h (sCRgi-24h).

It seemed possible that reducing insulin and glucose concentrations for <24 h might be sufficient to alter glucose transport. Accordingly, Protocol 5 was identical to Protocol 4, except that the duration of incubation step 1 was 6 h. The groups with glucose and insulin similar to AL and CR values were designated at sALgi-6h and sCRgi-6h, respectively.

Following incubation step 1, muscles from all protocols were rinsed in Krebs-Henseleit buffer supplemented with 0.1% BSA, 2 mM sodium pyruvate, and 5 mM mannitol for two 30-min periods (incubation steps 2 and 3) at 30°C. During steps 2 and 3 for all protocols, the Krebs-Henseleit buffer included either no insulin (basal) or 80 µU/ml insulin. The insulin concentration during steps 2 and 3 were identical for paired muscles (i.e., either both muscles from each rat were incubated without insulin or both with 80 µU/ml insulin).

Incubation with 3-MG. In the final incubation step (step 4) for every protocol, muscles were incubated at 30°C for 15 min in 1.5 ml of Krebs-Henseleit buffer containing 0.1% BSA, 8 mM 3-MG (including 0.25 mCi/mmol [3H]3-MG), and 2 mM mannitol (including 0.1 mCi/mmol [14C]mannitol). The insulin concentration remained the same as in steps 2 and 3 (0 or 80 µU/ml insulin). Muscles were then rapidly blotted, trimmed, clamp frozen with aluminum tongs cooled to the temperature of liquid N2, and stored at –80°C until further processing.

Muscle lysate preparation. Frozen muscles were weighed, transferred to prechilled glass tissue grinding tubes (Kontes, Vineland, NJ), and homogenized in ice-cold lysis buffer (1 ml/muscle) containing 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 (vol/vol), 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM sodium vanadate, 1 mM β-glycerophosphate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were transferred to microfuge tubes, rotated for 1–2 h at 4°C, and then centrifuged at 15,000 g for 15 min to remove insoluble material. Following homogenization, processed muscle samples were used for determination of glucose transport as well as ELISA and Western blotting. Duplicate 200-µl aliquots of the supernatants were used for scintillation counting. [3H]3-MG uptake was determined as previously described (10). Protein concentrations of the same supernatants were determined by the bicinchoninic acid method.

ELISA. IR abundance and IR tyrosine phosphorylation in muscle homogenates were measured using Total IR (no. KHR9111) and pY-IR (no. KHR9131) ELISA kits, respectively, according to the manufacturer's instructions (BioSource International).

Immunoblotting. Aliquots of supernatants (40–60 mg protein) were solubilized in SDS sample buffer, heated at 95°C for 3 min, and subjected to 10% SDS-PAGE. Resolved proteins were transferred to nitrocellulose paper overnight at a constant current of 100 mA/transfer apparatus in electrotransfer buffer (20 mM Tris, pH 8.0, 150 mM glycine, 0.025% SDS, and 20% methanol). Nitrocellulose blots were blocked in 5% milk in TBST (0.1% Tween 20 in Tris-buffered saline, pH 7.5) for 1 h at room temperature and transferred to 5% BSA-TBST with primary antibody for 3 h at room temperature or overnight at 4°C. Blots were then washed and incubated in anti-rabbit IgG horseradish peroxidase (1:20,000) for 1 h at room temperature. Blots were washed of excess antibody and subjected to ECL, and immunoreactive protein was quantified by densitometry.

Statistical analysis. All data are expressed as means ± SE. Differences between paired muscles were determined by paired t-test using SigmaSTAT (SPSS, Chicago, IL). P ≤ 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
3-MG transport. In Protocol 1, basal glucose transport was not different between muscles with an initial incubation period of 1 h compared with muscles undergoing a 24-h initial incubation step (data not shown). Glucose transport determined with insulin was ~31% greater (P < 0.05) for muscles in the sALgi-24h group compared with paired muscles in the sALgi-1h group (data not shown).

In Protocol 2, incubating muscles for 24 h with only glucose reduced (sCRg-24h) compared with controls (sALgi-24h) did not significantly alter subsequent glucose uptake measured with or without insulin (Fig. 2). Similarly, in Protocol 3, reducing only insulin for 24 h (sCRi-24h) did not alter subsequent glucose transport measured with or without insulin (Fig. 3). However, in Protocol 4, when both glucose and insulin were reduced for 24 h, subsequent glucose transport with insulin was increased ~29% in sCRgi-24h compared with sAL-24h (P < 0.005) muscles, whereas basal glucose transport was unaffected (Fig. 4). In Protocol 5, incubation with reduced glucose and insulin for 6 h (sCRgi-6h) compared with controls (sALgi-6h) was insufficient to alter glucose transport without or with insulin (data not shown).


Figure 2
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  		Fig. 2. Rate of 3-MG transport determined for epitrochlearis muscles in Protocol 2. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 80 µU/ml insulin (sCRg-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1 h before measurement of 3-MG transport. Data are means ± SE for 5 (basal) or 8 (insulin stimulated) muscles per group. There were no statistically significant differences between the sALgi-24h and sCRg-24h groups.

 

Figure 3
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  		Fig. 3. Rate of 3-MG transport determined for epitrochlearis muscles in Protocol 3. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 8 mM glucose + 20 µU/ml insulin (sCRi-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1 h before measurement of 3-MG transport. Data are means ± SE for 8 (basal) or 8 (insulin-stimulated) muscles per group. There were no statistically significant differences between the sALgi-24h and sCRi-24h groups.

 

Figure 4
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  		Fig. 4. Rate of 3-MG transport determined for epitrochlearis muscles in Protocol 4. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 20 µU/ml insulin (sCRgi-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1 h before measurement of 3-MG transport. Data are means ± SE for 15 (basal) or 14 (insulin-stimulated) muscles per group. *P < 0.05, 3-MG for insulin-stimulated muscles from the sALgi-24h vs. sCRgi-24h group.

 
ELISA. IR abundance was not significantly different between paired muscles incubated with both glucose and insulin reduced for 24 h (sCRgi-24h) and sALgi-24h controls (Fig. 5A). The glucose and insulin concentrations during the initial incubation step did not alter the subsequent level of IR tyrosine phosphorylation for sCRgi-24h compared with sALgi-24h groups under basal or insulin-stimulated conditions (Fig. 5B).


Figure 5
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  		Fig. 5. Insulin receptor (IR) abundance and tyrosine phosphorylated (pY) IR determined for epitrochlearis muscles in Protocol 4. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 20 µU/ml insulin (sCRgi-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1.25 h. A: there was no statistically significant difference between the sALgi-24h and sCRgi groups for IR abundance. Data are means ± SE for 26 muscles per group. B: there were no statistically significant differences for sALgi-24h vs. sCRgi groups for pY IR. Data are means ± SE for 12 (basal) or 14 (insulin stimulated) muscles per group.

 
Immunoblotting. Muscle IRS-1 abundance was not significantly different for muscles incubated for 24 h with both glucose and insulin reduced (sCRgi-24h) compared with sALgi-24h controls (data not shown). There were also no differences in pSerAkt (Fig. 6) or anti-PAS immunoreactivity at 160 kDa (PAS-160kDa; Fig. 7) between sALgi-24h and sCRgi-24h groups in muscles under basal or insulin-stimulated conditions. PAS immunoreactivity at 160 kDa is an indicator of the amount of phosphorylated Akt substrate of 160 kDa (AS160) (8, 27, 37). GLUT1 abundance was not different between the sCRgi-24h and sALgi-24h groups (data not shown). There also was not a significant difference in GLUT4 abundance in muscles from the sCRgi-24h compared with the sALgi-24h group (Fig. 8).


Figure 6
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  		Fig. 6. Akt serine phosphorylation [pAkt(S473)] determined for epitrochlearis muscles in Protocol 4. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 20 µU/ml insulin (sCRgi-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1.25 h. Data are means ± SE for 15 (basal) or 14 (insulin-stimulated) muscles per group. S473, Ser473. There were no statistically significant differences between the sALgi-24h and sCRgi-24h groups.

 

Figure 7
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  		Fig. 7. Phosphorylated protein of apparent molecular weight of 160 kDa identified using the PAS antibody (PAS-160kDa) determined for epitrochlearis muscles in Protocol 4. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 20 µU/ml insulin (sCRgi-24h) for 24 h. Subsequently, both muscles from each rat were incubated either without insulin or with 80 µU/ml insulin for 1.25 h. Data are means ± SE for 12 (basal) or 10 (insulin-stimulated) muscles per group. There were no statistically significant differences between the sALgi-24h and sCRgi-24h groups.

 

Figure 8
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  		Fig. 8. GLUT4 abundance was determined for epitrochlearis muscles in Protocol 4. Paired muscles were initially incubated with 8 mM glucose + 80 µU/ml insulin (sALgi-24h) or 5.5 mM glucose + 20 µU/ml insulin (sCRgi-24h) for 24 h. There was no statistically significant difference between the sALgi-24h and sCRgi-24h groups for GLUT4 abundance. Data are means ± SE for 29 muscles per group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The most important new findings of this study are the following: 1) exposure of isolated rat skeletal muscle for 24 h to moderate reductions in both extracellular glucose and insulin, comparable to changes found with in vivo CR, can trigger an increase in insulin-stimulated glucose transport with unaltered basal glucose transport; 2) reduction of only glucose or insulin was insufficient for this adaptation; 3) reduction of glucose and insulin for only 6 h was also insufficient for elevated insulin-stimulated glucose transport; 4) the increased glucose transport was not attributable to increased tyrosine phosphorylation of the insulin receptor or increased GLUT4 abundance; and 5) the mechanism for the improved glucose transport appeared to be distinct from that after in vivo CR, as pSerAkt of insulin-stimulated muscles was unaffected.

The absence of a significant effect of reducing extracellular glucose from 8 to 5.5 mM glucose on basal glucose transport in the present study is similar to the small or absent effects previously reported for intact skeletal muscle, in which the difference in glucose concentration was 5 vs. 10 mM (38). The present results extend the earlier findings because the duration of glucose exposure was considerably longer (24 compared with 3 h). Furthermore, unlike the earlier study (38), the present study included measurement of insulin-stimulated glucose transport, which was not found to differ for muscles with prolonged exposure at these levels of glucose. In the absence of changes in insulin concentration, there appears to be little effect on glucose transport when glucose is modulated within this physiological range, at least for glucose exposures of up to 24 h.

Glucose transport was also unaltered after 24-h incubation with 20 compared with 80 µU/ml insulin in the presence of 8 mM glucose. These results also indicate that, in the presence of a normal extracellular glucose concentration, a 75% reduction in insulin concentration within the physiological range did not modify subsequent glucose transport. These findings are in contrast to data from rat hindlimbs perfused for 2 h with 5 mM glucose and 50 vs. 20,000 µU/ml insulin (26). Under those conditions, prior perfusion with supraphysiological compared with physiological insulin was characterized by reduced glucose uptake. Alterations in muscle glucose uptake after exposure to extreme differences in insulin concentration (no insulin vs. physiological insulin levels or physiological vs. supraphysiological insulin levels) cannot be considered predictive of the outcome after prolonged exposure to differing insulin concentrations within the normal physiological range.

Reductions in both glucose and insulin were required for the elevated insulin-stimulated glucose transport. One interpretation of the requirement for lowering both glucose and insulin concentrations to achieve increased insulin sensitivity is that the mechanism is not simply a direct effect of reduced insulin signaling but rather is secondary, at least in part, to reduced insulin-stimulated glucose flux into the cell because of decrements in both glucose and insulin concentrations.

The prolonged incubation with reduced glucose and insulin concentrations recapitulated the effect of in vivo CR on glucose transport, i.e., glucose transport was elevated in skeletal muscle stimulated with submaximally effective insulin, and basal glucose transport was unaltered. The magnitude of the increase in glucose transport observed (29% above controls) compares with increments of ~20–100% in earlier studies of isolated epitrochlearis muscles from in vivo CR vs. ad libitum-fed rats (11, 12, 15–17, 33, 34). Thus the relative magnitude of the adaptation with the present model overlaps with the lower range of effects reported after in vivo CR.

Although the change in glucose transport was similar with the in vitro model compared with earlier studies with in vivo CR, the lack of an increase in Akt phosphorylation differs from our previous finding that in vivo CR leads to increased Akt phosphorylation in rodent muscle stimulated with submaximally effective insulin concentrations (32–34). Data from mice that are null for Akt2, the isoform that is important for insulin-stimulated glucose transport (6), indicate that Akt2 is essential for the full effect of in vivo CR on insulin-stimulated glucose transport in skeletal muscle (32). AS160 is the most distal insulin signaling protein that is known to link insulin's activation of Akt to GLUT4 translocation (9, 13, 27, 28, 37). The lack of a difference in PAS-160kDa for the sALgi-24h compared with sCRgi-24h groups is consistent with the observation of no difference in Akt phosphorylation between these groups. The effect of in vivo CR on insulin-stimulated AS160 phosphorylation has not been reported. However, because in vivo CR leads to an increase in insulin-stimulated Akt phosphorylation (32–34), it seems reasonable to hypothesize that AS160 phosphorylation may be greater for insulin-stimulated muscles from CR compared with ad libitum-fed rats.

Previous research using Akt2-null mice suggested that improved Akt2 phosphorylation was not likely the only mechanism for improved insulin sensitivity after in vivo CR (32). The Akt2-independent mechanism whereby CR can improve insulin sensitivity has not been identified, but it is conceivable that there is an enhancement in an insulin signaling step that has not yet been evaluated with CR, e.g., atypical protein kinase C. Another possibility is that there is a modification of the molecular machinery that performs GLUT4 trafficking downstream of the insulin signaling cascade. Regardless, it is unclear whether the Akt-independent mechanism with the present model is also playing any role in the improved insulin action found with in vivo CR.

Previous research that quantified insulin receptors in skeletal muscle using various methods, including Western blotting (2–4, 42) and insulin receptor binding (7, 14), has usually found no effect of CR, consistent with the present results, although Wang et al. (39) reported increased insulin receptor binding in rat skeletal muscle after CR. Two studies have indicated that insulin receptor tyrosine phosphorylation can be increased by CR in muscle (3, 18), whereas an equal number of studies have not found this effect of CR (2, 4). In addition, Zhu et al. (42, 43) found that CR led to increased insulin receptor tyrosine phosphorylation in muscle from old, but not young, rats. Thus the lack of an increase in insulin receptor tyrosine phosphorylation after exposure to reduced glucose and insulin is consistent with some, but not all, of the published effects of in vivo CR on insulin receptor tyrosine phosphorylation. We previously found that in vivo CR can lead to a reduction in rat and mouse skeletal muscle IRS1 abundance (16, 21, 34), although others did not find this result in mouse skeletal muscle (2–4), and we did not find this CR effect in muscle from nonhuman primates (22). In any case, a reduction in muscle IRS1 levels would not be expected to account for improved insulin sensitivity. GLUT4 abundance was not significantly altered in the muscles after prolonged incubation with reduced glucose and insulin. These results are consistent with most earlier research on in vivo CR, which indicates no effect of CR on skeletal muscle GLUT4 total abundance (2, 4, 12, 22, 39), although one previous study reported an increase in mouse muscle GLUT4 with CR (3). GLUT1 abundance was unaffected in the present study, consistent with the lack of an effect of in vivo CR on GLUT1 from rat soleus or white gastrocnemius muscles, although a CR-associated increase in GLUT1 was found in a mixed sample of whole gastrocnemius and quadriceps muscles (40). In skeletal muscle, GLUT1 is involved in basal rather than insulin-stimulated glucose transport, so the lack of altered GLUT1 abundance is consistent with the unaltered basal glucose transport in this model and with in vivo CR.

In conclusion, this study provides the proof of principle that moderate reductions in extracellular glucose and insulin, similar to those found with in vivo CR, are sufficient to induce an increase in insulin-stimulated glucose transport of muscle that is comparable to the increase found with in vivo CR. The results for insulin receptor, GLUT1, and GLUT4 abundance after incubating muscles with reduced extracellular glucose and insulin were consistent with the findings of most studies of in vivo CR. However, the absence of an increase in Akt phosphorylation in this in vitro model, as opposed to the increased Akt phosphorylation that has usually been observed with in vivo CR, suggests that the mechanisms for improved insulin sensitivity are distinct for the two conditions. The present study was an initial approach in which only extracellular glucose and insulin were manipulated. Modifying the model to include other CR-induced effects (e.g., a decrement in extracellular adiponectin), simulation of diurnal modulations of glucose and insulin, and extension of the experiment beyond 24 h would likely provide further insights into the mechanisms for improved insulin sensitivity with in vivo CR.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institute on Aging Grants AG-10026 and AG-024824.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. B. Arias, Division of Kinesiology, Univ. of Michigan, 401 Washtenaw Ave., Ann Arbor, MI 48109 (e-mail: edarias{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Arciero PJ, Vukovich MD, Holloszy JO, Racette SB, Kohrt WM. Comparison of short-term diet and exercise on insulin action in individuals with abnormal glucose tolerance. J Appl Physiol 86: 1930–1935, 1999.[Abstract/Free Full Text]
  2. Argentino DP, Dominici FP, Al-Regaiey K, Bonkowski MS, Bartke A, Turyn D. Effects of long-term caloric restriction on early steps of the insulin-signaling system in mouse skeletal muscle. J Gerontol A Biol Sci Med Sci 60: 28–34, 2005.[Abstract/Free Full Text]
  3. Argentino DP, Dominici FP, Munoz MC, Al-Regaiey K, Bartke A, Turyn D. Effects of long-term caloric restriction on glucose homeostasis and on the first steps of the insulin signaling system in skeletal muscle of normal and Ames dwarf (Prop1df/Prop1df) mice. Exp Gerontol 40: 27–35, 2005.[CrossRef][Web of Science][Medline]
  4. Argentino DP, Munoz MC, Rocha JS, Bartke A, Turyn D, Dominici FP. Short-term caloric restriction does not modify the in vivo insulin signaling pathway leading to Akt activation in skeletal muscle of Ames dwarf (Prop1(df)/Prop1(df)) mice. Horm Metab Res 37: 672–679, 2005.[CrossRef][Web of Science][Medline]
  5. Arias EB, Kim J, Cartee GD. Prolonged incubation in PUGNAc results in increased protein O-linked glycosylation and insulin resistance in rat skeletal muscle. Diabetes 53: 921–930, 2004.[Abstract/Free Full Text]
  6. Bae SS, Cho H, Mu J, Birnbaum MJ. Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J Biol Chem 278: 49530–49536, 2003.[Abstract/Free Full Text]
  7. Balage M, Grizard J, Manin M. Effect of calorie restriction on skeletal muscle and liver insulin binding in growing rat. Horm Metab Res 22: 207–214, 1990.[Web of Science][Medline]
  8. Bjornholm M, He AR, Attersand A, Lake S, Liu SC, Lienhard GE, Taylor S, Arner P, Zierath JR. Absence of functional insulin receptor substrate-3 (IRS-3) gene in humans. Diabetologia 45: 1697–1702, 2002.[CrossRef][Web of Science][Medline]
  9. Bruss MD, Arias EB, Lienhard GE, Cartee GD. Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity. Diabetes 54: 41–50, 2005.[Medline]
  10. Cartee GD, Bohn EE. Growth hormone reduces glucose transport but not GLUT-1 or GLUT-4 in adult and old rats. Am J Physiol Endocrinol Metab 268: E902–E909, 1995.[Abstract/Free Full Text]
  11. Cartee GD, Dean DJ. Glucose transport with brief dietary restriction: heterogenous responses in muscles. Am J Physiol Endocrinol Metab 266: E946–E952, 1994.[Abstract/Free Full Text]
  12. Cartee GD, Kietzke EW, Briggs-Tung C. Adaptation of muscle glucose transport with caloric restriction in adult, middle-aged, and old rats. Am J Physiol Regul Integr Comp Physiol 266: R1443–R1447, 1994.[Abstract/Free Full Text]
  13. Cartee GD, Wojtaszewski JF. Role of Akt substrate of 160 kDa in insulin-stimulated and contraction-stimulated glucose transport. Appl Physiol Nutr Metab 32: 557–566, 2007.[CrossRef][Web of Science][Medline]
  14. Cecchin F, Ittoop O, Sinha MK, Caro JF. Insulin resistance in uremia: insulin receptor kinase activity in liver and muscle from chronic uremic rats. Am J Physiol Endocrinol Metab 254: E394–E401, 1988.[Abstract/Free Full Text]
  15. Davidson RT, Arias EB, Cartee GD. Calorie restriction increases muscle insulin action but not IRS-1-, IRS-2-, or phosphotyrosine-PI 3-kinase. Am J Physiol Endocrinol Metab 282: E270–E276, 2002.[Abstract/Free Full Text]
  16. Dean DJ, Brozinick JT Jr, Cushman SW, Cartee GD. Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle. Am J Physiol Endocrinol Metab 275: E957–E964, 1998.[Abstract/Free Full Text]
  17. Dean DJ, Cartee GD. Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. J Gerontol A Biol Sci Med Sci 51: B208–B213, 1996.[Abstract]
  18. Dean DJ, Cartee GD. Calorie restriction increases insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 in rat skeletal muscle. Acta Physiol Scand 169: 133–139, 2000.[CrossRef][Web of Science][Medline]
  19. DeFronzo RA. Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37: 667–687, 1988.[Web of Science][Medline]
  20. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 86: 3574–3578, 2001.[Abstract/Free Full Text]
  21. Gazdag AC, Dumke CL, Kahn CR, Cartee GD. Calorie restriction increases insulin-stimulated glucose transport in skeletal muscle from IRS-1 knockout mice. Diabetes 48: 1930–1936, 1999.[Abstract]
  22. Gazdag AC, Sullivan S, Kemnitz JW, Cartee GD. Effect of long-term caloric restriction on GLUT4, phosphatidylinositol-3 kinase p85 subunit, and insulin receptor substrate-1 protein levels in rhesus monkey skeletal muscle. J Gerontol A Biol Sci Med Sci 55: B44–B46; discussion B47–B48, 2000.[Abstract]
  23. Gazdag AC, Wetter TJ, Davidson RT, Robinson KA, Buse MG, Yee AJ, Turcotte LP, Cartee GD. Lower calorie intake enhances muscle insulin action and reduces hexosamine levels. Am J Physiol Regul Integr Comp Physiol 278: R504–R512, 2000.[Abstract/Free Full Text]
  24. Gulve EA, Cartee GD, Holloszy JO. Prolonged incubation of skeletal muscle in vitro: prevention of increases in glucose transport. Am J Physiol Cell Physiol 261: C154–C160, 1991.[Abstract/Free Full Text]
  25. Gupta G, She L, Ma XH, Yang XM, Hu M, Cases JA, Vuguin P, Rossetti L, Barzilai N. Aging does not contribute to the decline in insulin action on storage of muscle glycogen in rats. Am J Physiol Regul Integr Comp Physiol 278: R111–R117, 2000.[Abstract/Free Full Text]
  26. Hansen BF, Hansen SA, Ploug T, Bak JF, Richter EA. Effects of glucose and insulin on development of impaired insulin action in muscle. Am J Physiol Endocrinol Metab 262: E440–E446, 1992.[Abstract/Free Full Text]
  27. Kane S, Sano H, Liu SC, Asara JM, Lane WS, Garner CC, Lienhard GE. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J Biol Chem 277: 22115–22118, 2002.[Abstract/Free Full Text]
  28. Kramer HF, Witczak CA, Taylor EB, Fujii N, Hirshman MF, Goodyear LJ. AS160 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. J Biol Chem 281: 31478–31485, 2006.[Abstract/Free Full Text]
  29. Masoro EJ, Katz MS, McMahan CA. Evidence for the glycation hypothesis of aging from the food-restricted rodent model. J Gerontol 44: B20–B22, 1989.[Abstract]
  30. Masoro EJ, McCarter RJ, Katz MS, McMahan CA. Dietary restriction alters characteristics of glucose fuel use. J Gerontol 47: B202–B208, 1992.[Abstract]
  31. Mayor P, Maianu L, Garvey WT. Glucose and insulin chronically regulate insulin action via different mechanisms in BC3H1 myocytes. Effects on glucose transporter gene expression. Diabetes 41: 274–285, 1992.[Abstract]
  32. McCurdy CE, Cartee GD. Akt2 is essential for the full effect of calorie restriction on insulin-stimulated glucose uptake in skeletal muscle. Diabetes 54: 1349–1356, 2005.[Abstract/Free Full Text]
  33. McCurdy CE, Davidson RT, Cartee GD. Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle. Am J Physiol Endocrinol Metab 285: E693–E700, 2003.[Abstract/Free Full Text]
  34. McCurdy CE, Davidson RT, Cartee GD. Calorie restriction increases the ratio of phosphatidylinositol 3-kinase catalytic to regulatory subunits in rat skeletal muscle. Am J Physiol Endocrinol Metab 288: E996–E1001, 2005.[Abstract/Free Full Text]
  35. Muzumdar R, Ma X, Atzmon G, Vuguin P, Yang X, Barzilai N. Decrease in glucose-stimulated insulin secretion with aging is independent of insulin action. Diabetes 53: 441–446, 2004.[Abstract/Free Full Text]
  36. Novelli M, Masiello P, Bombara M, Bergamini E. Protein glycation in the aging male Sprague-Dawley rat: effects of antiaging diet restrictions. J Gerontol A Biol Sci Med Sci 53: B94–B101, 1998.[Abstract]
  37. Sano H, Kane S, Sano E, Miinea CP, Asara JM, Lane WS, Garner CW, Lienhard GE. Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J Biol Chem 278: 14599–14602, 2003.[Abstract/Free Full Text]
  38. Sasson S, Cerasi E. Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture. J Biol Chem 261: 16827–16833, 1986.[Abstract/Free Full Text]
  39. Wang ZQ, Bell-Farrow AD, Sonntag W, Cefalu WT. Effect of age and caloric restriction on insulin receptor binding and glucose transporter levels in aging rats. Exp Gerontol 32: 671–684, 1997.[CrossRef][Web of Science][Medline]
  40. Wilkes JJ, Nagy LE. Chronic ethanol feeding impairs glucose tolerance but does not produce skeletal muscle insulin resistance in rat epitrochlearis muscle. Alcohol Clin Exp Res 20: 1016–1022, 1996.[CrossRef][Web of Science][Medline]
  41. Young DA, Uhl JJ, Cartee GD, Holloszy JO. Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations. J Biol Chem 261: 16049–16053, 1986.[Abstract/Free Full Text]
  42. Zhu M, de Cabo R, Anson RM, Ingram DK, Lane MA. Caloric restriction modulates insulin receptor signaling in liver and skeletal muscle of rat. Nutrition 21: 378–388, 2005.[CrossRef][Web of Science][Medline]
  43. Zhu M, Miura J, Lu LX, Bernier M, DeCabo R, Lane MA, Roth GS, Ingram DK. Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization. Exp Gerontol 39: 1049–1059, 2004.[CrossRef][Web of Science][Medline]




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