Fiber Type–Specific Differences in Glucose Uptake by Single Fibers From Skeletal Muscles of 9- and 25-Month-Old Rats

James G MacKrell; Edward B Arias; Gregory D Cartee

doi:10.1093/gerona/gls194

. 2012 Oct 4;67(12):1286–1294. doi: 10.1093/gerona/gls194

Fiber Type–Specific Differences in Glucose Uptake by Single Fibers From Skeletal Muscles of 9- and 25-Month-Old Rats

James G MacKrell ^1,², Edward B Arias ¹, Gregory D Cartee ^1,^2,^3,^✉

PMCID: PMC3670157 PMID: 23042591

Abstract

The primary purpose of this study was to assess the feasibility of applying a novel approach to measure myosin heavy chain (MHC) isoform expression, glucose uptake, fiber volume, and protein abundance in single muscle fibers of adult (9 months) and old (25 months) rats. Epitrochlearis muscle fibers were successfully isolated and analyzed for MHC isoform expression, glucose uptake, fiber volume, and protein (COXIV, APPL1, IκB-β) abundance. Insulin-stimulated glucose uptake by single fibers did not differ between age groups, but there was a significant difference between fiber types (IIA > IIX > IIB/X ≈ IIB). There were also significant main effects of fiber type on APPL1 (IIX > IIB) and COXIV (IIA > IIX > IIB/X ≈ IIB) abundance, and IIB fibers were significantly larger than IIA fibers. This study established the feasibility of a new approach for assessing age-related differences in muscle at the single-fiber level and demonstrated the magnitude and rank order for fiber-type differences in insulin-stimulated glucose uptake of 9-month-old and 25-month-old rats.

Key Words: Myosin heavy chain, Insulin resistance, Insulin sensitivity, Glucose transport, Sarcopenia

Many of the most prevalent and devastating age-related diseases in humans (including hypertension, coronary heart disease, stroke, some forms of cancer, and type 2 diabetes) have been linked to insulin resistance (1). Whole-body glucose disposal during a euglycemic-hyperinsulinemic clamp has been reported to be moderately (~15%–35%) lower for old rats (20 to 25 months old) compared with adult rats (6 to 10 months old) (2,3). Skeletal muscle is the major tissue for insulin-mediated glucose clearance, but the magnitude of age-related insulin resistance does not appear to be uniform in all skeletal muscles. Previous studies have compared 8- to 9-month-old and 24- to 25-month-old rats for glucose uptake by multiple muscles under in vivo conditions (during euglycemic-hyperinsulinemic clamp) (4) and under ex vivo conditions (in isolated muscles) (5). In both conditions, relatively greater age-related decrements in glucose uptake were reported for skeletal muscles that were mostly composed of type I and type IIA fibers, and lesser levels of age-related insulin resistance in muscles that were largely composed of type IIB and IIX fibers. However, it is uncertain if the variable extent of age-related insulin resistance is solely attributable to differences in fiber-type composition or if there may be a contribution of other differences between the particular skeletal muscles that have been studied. In this context, it would be informative to measure glucose uptake by muscle fibers of differing fiber types from the same muscle.

There is striking diversity in fiber-type composition between different muscles from the same animal. Furthermore, each skeletal muscle is composed of hundreds or thousands of muscle fibers that can also be diverse in fiber type and metabolic characteristics. We recently developed and validated the first method allowing for the isolation of single mammalian skeletal muscle fibers to be used for measurement of fiber type, fiber size, and glucose uptake (6). Using isolated epitrochlearis muscles from 2- to 3-month-old rats, we found fiber type–dependent differences in insulin-stimulated glucose uptake. Fibers expressing the IIA myosin heavy chain (MHC) isoform had significantly greater glucose uptake than all other fiber types measured (IIA > IIB, IIX, IIB/X). Furthermore, our novel single-fiber method provided the first information about the glucose uptake capacity of hybrid fibers (expressing more than one MHC isoform; IIB/X) (6) that have been reported to account for ~5% to 30% of fibers in some rodent muscles (7).

In a series of studies using isolated whole epitrochlearis muscles from adult (8 to 13 months old) compared with old (23 to 31 months old), we have found age-related differences in insulin-stimulated glucose uptake ranging from 0% to 18% (8–11). It seemed possible that the small fraction of highly oxidative fibers in this muscle [including ~6%–13% type IIA fibers and ~6%–8% type I fibers ) (12,13) may account for a disproportionate amount of the modest age-related insulin resistance that has sometimes been detected in the whole muscle. In our earlier study using 2- to 3-month-old rats, we were able to isolate and measure glucose uptake of single fibers expressing type IIA, IIB, IIX, and IIB/X MHC (6). Accordingly, this new method offered the opportunity to evaluate the idea that the extent of age-related insulin resistance might be variable among these fiber types.

The overall goal of the current study was to advance the currently limited understanding of the relationship between fiber type and age-associated insulin resistance in skeletal muscle by studying muscle glucose uptake of adult and old rats for the first time at the level of the single fiber. The first aim was to determine whether our novel single-fiber method for measuring glucose uptake and MHC isoform expression could be successfully applied to epitrochlearis muscles from adult (9 months old) and old (25 months old) rats. The second aim was to determine whether insulin-stimulated glucose uptake differs among fiber types in either adult or old rats. The third aim was to determine whether the extent of age-associated insulin resistance for glucose uptake is fiber-type dependent. Our final aim was to determine possible age and fiber-type differences for expression of selected proteins: cytochrome C oxidase subunit IV (COXIV, mitochondrial electron transport chain protein), IκB-β (marker of IκB/Nuclear factor-κB, NFκB, pathway activation), and Appl1 (an adapter protein involved in mediating signaling and metabolic effects of adiponectin).

Research Design and Methods

Materials

Human recombinant insulin was from Eli Lilly (Indianapolis, IN). Bicinchoninic acid (BCA) protein assay reagents and T-PER reagent were from Pierce Biotechnology (Rockford, IL). [³H]-2-Deoxy-D-glucose ([³H]-2-DG) was from Perkin Elmer (Waltham, MA). Collagenase (type II) was from Worthington Biochem Corp. (Lakewood, NJ). Trypan Blue was from Invitrogen (Carlsbad, CA). Reagents and apparatus for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were purchased from Bio-Rad (Hercules, CA). West Dura Extended Duration Substrate was from Pierce Biotechnology. Anti-APPL1 antibody (#ab59592) and anti-COXIV (#ab16506) were from Abcam (Cambridge, MA); anti-IκB-β antibody (#sc-945) was from Santa Cruz (Santa Cruz, CA); and anti-rabbit IgG horseradish peroxidase (#7074) was from Cell Signaling Technology (Danvers, MA). Other reagents were from Sigma-Aldrich (St Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Animal Treatment

Procedures for animal care were approved by the University of Michigan Committee on Use and Care of Animals. Male Fisher-344 × Brown Norway, F1 generation rats were obtained at 9 or 25 months from Harlan (Indianapolis, IN). Animals were provided with rodent chow ad libitum until 5 PM the night before the experiment, when food was removed. The next day, between 10 AM and 1 PM, rats were anesthetized (intraperitoneal injection of sodium pentobarbital), and both epitrochlearis muscles were extracted.

Muscle Incubation

Isolated muscles underwent a series of incubation steps as previously described (6). In brief, isolated muscles were incubated in vials containing 2mL of Krebs Henseleit buffer (KHB) supplemented with 0.1% bovine serum albumin (KHB-BSA), 2mM sodium pyruvate, and 6mM mannitol with 0 or 12nM insulin for 20 minutes, and then transferred to a second vial for 60-minute incubation in 2mL of KHB-BSA, 1mM 2-DG (specific activity of 13.5 mCi/mmol [³H]-2-DG), and 9mM mannitol and the same insulin concentration as the previous step. Muscles underwent 3x5-minute washes (100rpm) in ice-cold KHB-BSA to clear the extracellular space of 2-DG (14), followed by incubation in Collagenase Media (Ca²⁺-free KHB and 1.5% type II collagenase) for 60 minutes (collagenase-treated muscles are hereafter referred to as fiber bundles). Unless otherwise noted, vials were shaken (45rpm), gassed (95% O₂/5% CO₂), and maintained at 35°C in a water bath.

Single-Fiber Isolation and Processing

Following collagenase incubation, fiber bundles were removed from solution and single fibers were isolated as previously described (6). In brief, single fibers were teased from the fiber bundle using forceps and imaged using a camera-enabled microscope with Leica Application Suite EZ software after isolation. Fiber width (mean value for width measured at three locations per fiber: near the fiber midpoint and approximately halfway between midpoint and each end of the fiber) and length of each fiber were used to estimate volume (V = πr ² l; r = fiber radius as determined by half of the width measurement, l = fiber length). Each fiber was transferred by pipette with 10 μL of solution to a microcentrifuge tube containing 40 μL of lysis buffer (T-PER, 1mM ethylenediaminetetraacetic acid, 1mM ethylene glycol tetraacetic acid, 2.5mM sodium pyrophosphate, 1mM Na₃VO₄, 1mM β-glycerophosphate, 1 μg/mL leupeptin, and 1mM phenylmethylsulfonyl fluoride). Laemmli buffer (2×, 50 μL) was added to each tube.

Single-Fiber 2-DG Uptake, MHC Isoform Characterization, and Immunoblotting

Separate aliquots of a lysed fiber were used for 2-DG uptake, MHC characterization, and protein abundance (immunoblotting) for single fibers as previously described (6). In brief, an aliquot of lysed fiber was used to determine [³H]-2-DG disintegrations per minute using a scintillation counter. Measured 2-DG accumulation was normalized to calculated fiber volume and expressed as nanomoles per microliter (nmol/µl). A separate aliquot of fiber lysate was used to determine MHC isoform expression via SDS-PAGE. Using the relative abundance of MHC expressed per fiber, a similar amount of MHC protein for each fiber aliquot was loaded onto 9% SDS-PAGE gels for subsequent immunoblotting and measurements of COXIV, Appl1, and IκB-β content as previously described (6). After immunoblotting, membranes were washed and subjected to enhanced chemiluminescence (West Dura Extended Duration Substrate; #34075; Pierce) to visualize protein bands. Immunoreactive proteins were quantified by densitometry (AlphaEase FC; Alpha Innotech, San Leandro, CA). Values for protein abundance of single fibers were normalized to the average of the adult (9 months old) samples on each blot and expressed relative to the fiber’s respective posttransfer MHC density determined in the gel (6).

Fiber Bundle Homogenization, 2-DG Uptake, and Immunoblotting

Following fiber isolation, the donor fiber bundles from which fibers were isolated were processed for 2-DG uptake and immunoblotting in 1mL ice-cold lysis buffer using glass-on-glass grinding tubes (6). Homogenates were rotated (4°C, 1 hour) before being centrifuged (12,000g, 10 minutes, 4°C). A portion of supernatant was used to determine protein concentration (BCA protein assay). Aliquots of supernatant used for 2-DG uptake were pipetted into vials for scintillation counting, and 2-DG uptake was determined and normalized to protein content (micromoles per microgram protein for fiber bundles) as previously described (6). Immunoblotting was performed as previously described (5). Values are expressed relative to the normalized average of the adult (9 months old) samples on each blot. Remaining supernatant was stored at −80°C until further analysis.

Statistical Analysis

Data are expressed as mean ± SEM. Two-way ANOVA was used to identify significant main effects (age and fiber type) and interactions for glucose uptake, fiber size (estimated volume), and protein abundance (Appl1, IκB-β, and COXIV) in single fibers. Tukey’s post hoc t test was applied to determine the source of significant variance. Student’s t test was used to identify age differences for body mass, fiber bundle protein abundance, and fiber-type composition. A p value of lesser than or equal to .05 was considered statistically significant.

Results

Body Mass, Epitrochlearis MHC Composition, and Estimated Fiber Volume

Body mass was 18% greater (p < .05) in old (530.2±11.6g) versus adult (432.4±7.2g) rats. The relative abundance of each MHC isoform (I, IIB, IIX, and IIA) in the whole epitrochlearis muscles did not differ between adult (type I = 10.5% ± 0.6, IIB = 42.3% ± 3.4, IIX = 34.8% ± 2.0, IIA = 12.3% ± 1.2) and old (type I = 8.7% ± 0.7, IIB = 40.9% ± 1.8, IIX = 39.4% ± 2.2, IIA = 11.0% ± 1.1) rats. The volume of each isolated single fiber was estimated using width and length measurements. There was no significant effect of age on fiber volume, but there was a significant main effect of fiber type on fiber volume. Post hoc analysis revealed that the volume of IIB fibers was significantly greater (p < .05) than the volume of IIA fibers from adult and old rat muscle (Figure 1).

Figure 1. — Size (volume, microliter) of single muscle fibers expressing the same MHC isoform from adult (open bars) versus old (closed bars) epitrochlearis muscles. For fibers from adult rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB (109), IIA(19), IIX(18), IIB/X (54). For fibers from old rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB (103), IIA(11), IIX(11), IIB/X(35). Data are means ± *SEM* and were analyzed by two-way ANOVA. AGE, main effect of age (9 months vs. 25 months); FT, main effect of fiber type; AGExFT, interaction between main effects. There was a main effect of fiber type (p < .05). MHC isoforms that were revealed by post hoc analysis to be significantly different from each other (p < .05 for IIB vs. IIA) are designated by not having the same letter above their respective bars, whereas isoforms that were not significantly different are designated by having the same letter above their respective bars.

2-DG Uptake by Fiber Bundles and Single Fibers

No significant effect of age was detected for 2-DG uptake by the “donor” fiber bundles (from which single fibers had been previously isolated) incubated either in the absence or in the presence of insulin (Figure 2A). Basal (no insulin) 2-DG uptake values by single fibers were not significantly different between adult and old rats within any of the fiber types (Figure 2B). There was also no significant age-related difference for insulin-stimulated 2-DG uptake within any of the fiber types. However, in single fibers from insulin-stimulated muscles, there was a significant main effect of fiber type (p < .001) on 2-DG uptake. Post hoc analysis revealed that for single fibers from insulin-stimulated muscles (either adult or old), 2-DG uptake was greater for IIA fibers compared with all other fiber types (IIA > IIB, IIX, and IIB/X; p < .001). In addition, 2-DG uptake was significantly greater (p < .05) for IIX versus IIB fibers from insulin-stimulated muscles.

**. (A)** Basal and insulin-stimulated 2-DG uptake by adult (open bars) versus old (closed bars) fiber bundles (from which single fibers had been previously isolated). Data are means ± *SEM* (n = 5–6 muscles per age group at each insulin concentration). (B) Mean 2-DG uptake by single muscle fibers expressing the same MHC isoform from adult (open bars) versus old (closed bars) epitrochlearis muscles. For basal fibers, from adult rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB (56), IIA(5), IIX(9), IIB/X(20). For basal fibers, from old rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB(46), IIA(4), IIX(5), IIB/X(15). For insulin-stimulated fibers, from adult rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB (53), IIA(14), IIX(9), IIB/X(34). For insulin-stimulated fibers, from old rat muscle, the number of fibers for each MHC isoform is in parentheses: IIB (57), IIA(7), IIX(6), IIB/X(20). Data are means ± *SEM* and were analyzed by two-way ANOVA within each insulin level. AGE, main effect of age (9 months vs. 25 months); FT, main effect of fiber type; AGExFT, interaction between main effects. For fibers with insulin, there was a main effect of fiber type (p < .001). MHC isoforms that were significantly different from each other (p < .001 for IIA vs. IIB, IIX, and IIB/X; and p < .05 for IIX vs. IIB) are designated by not having the same letter above their respective bars, whereas isoforms that were not significantly different from each other are designated by having the same letter above their respective bars.

COXIV Abundance in Fiber Bundles and Single Fibers

In fiber bundles, there was no significant effect of age on COXIV abundance (Figure 3A). In single fibers, there were significant main effects of age (p < .05) and fiber type (p < .001; Figure 3B). Post hoc analysis revealed that COXIV content in IIA fibers was significantly greater (p < .001) than in IIB, IIX, and IIB/X fibers (IIA > IIB, IIX IIB/X; Figure 3B) and that COXIV content in IIX fibers was significantly greater (p < .01) than in IIB and IIB/X fibers (IIX > IIB and IIB/X; Figure 3B).

Appl1 Abundance in Fiber Bundles and Single Fibers

In fiber bundles, there was no significant effect of age on Appl1 abundance (Figure 4A). In single fibers, there was no main effect of age on Appl1 abundance, but there was significant main effect of fiber type (p < .01; Figure 4B). Post hoc analysis revealed the source of significant differences (p < .05) for APPL1 abundance among fiber types was IIX fibers versus IIB fibers (IIX > IIB).

IκB-β Abundance in Fiber Bundles and Single Fibers

In fiber bundles, there was no significant effect of age on IκB-β abundance (Figure 5A). There were also no significant effects of either age or fiber type on IκB-β abundance in single fibers (Figure 5B).

Discussion

A major outcome of the current study was demonstrating the feasibility of measuring glucose uptake, MHC isoform expression, estimated fiber volume, and protein abundance in single fibers of adult (9 months old) and old (25 months old) rats. Another significant and novel result was that in single fibers from both age groups, the rank order for insulin-stimulated glucose uptake by single fibers was found to be IIA > IIX > IIB/X ≈ IIB. The magnitude of fiber type–related differences for insulin-stimulated glucose uptake of adult and old rats was striking with values for type IIA fibers found to be more than twofold greater than IIX fibers and more than three- to fourfold greater than type IIB or IIB/X fibers from the same muscle. An additional valuable finding was the lack of age-related differences for insulin-stimulated glucose uptake by single fibers within any of the type II fiber types. There were also no age-related differences for MHC isoform content of whole muscles or for abundance of Appl1 and IκB-β at either the whole muscle level or within any of the fiber types. COXIV protein abundance was greater in both IIA and IIX fibers versus both IIB and IIB/X fibers in each age group. As expected, estimated fiber volume for type IIB fibers exceeded type IIA fibers in both age groups. No significant age-related differences in fiber volume were identified for any of the fiber types. Taken together, the current data demonstrate substantial fiber type–related differences for insulin-stimulated glucose uptake, COXIV protein abundance, and fiber volume.

Because of the phenotypic heterogeneity of different fiber types, it is essential to evaluate single fibers identified by fiber type to understand skeletal muscle at the cellular level. Previous studies have characterized single fibers from old individuals with regard to contractile properties, morphology, enzymatic activity, MHC expression, and mitochondrial deletions (15–23). However, the influence of aging on single-fiber glucose uptake was unknown because there was previously no method to measure glucose uptake of mammalian single fibers. The results of the current study demonstrated that insulin-stimulated glucose uptake measured at the single-fiber level within any type II MHC isoform is not lower for 25-month-old rats compared with 9-month-old rats. Earlier studies found that age-related insulin resistance in whole muscles appeared to be greater for muscles with a high abundance of type I and IIA fibers compared with muscles that were largely composed of fibers with high abundance of type IIB and IIX fibers (4,5). Therefore, we expected that we might find age-related insulin resistance in type IIA fibers, but the data did not support this expectation. It remains possible that type I fibers may be susceptible to age-related insulin resistance. However, the data from the current study do not provide direct information about this idea because, as in our earlier study of younger (2- to 3-month-old rats) (6), no type I fibers were isolated. We are unsure of the reason for the lack of type I fiber isolation, but it may be related to the higher level of collagenase that is reported to surround type I versus type II fibers (24). In support of this idea, despite multiple attempts to use collagenase with isolated soleus muscles (composed of approximately 90% type I fibers), we were unable to isolate type I fibers. Regardless, the protocol was successful in isolating single-fibers that expressed each of the other MHC isoforms (representing more than 90% of MHC expressed by the epitrochlearis) in proportions that were similar to their respective abundance in whole epitrochlearis (13,25).

Significant age-related differences in glucose uptake were not detected in either whole muscles or single fibers. We evaluated the influence of age on insulin-stimulated glucose uptake by the whole epitrochlearis muscle in several previous studies. We recently found a small (18%) reduction in glucose uptake of the whole epitrochlearis of 25- versus 9-month-old rats (5), but in several earlier studies of old (23 to 31 months old) compared with adult (8 to 13 months old) rats, similar to the results of the current study, we did not detect significant age-related differences in glucose uptake by the whole epitrochlearis (8–10). These results indicate that there is not profound insulin resistance in the whole epitrochlearis of rats across this portion of the adult life span. It is notable that in many rodents with experimentally introduced genetic modifications (ie, mice that express a mutated protein or that are null for expression of particular protein), insulin sensitivity can be normal under usual experimental conditions (eg, eating a standard rodent chow diet), with insulin resistance revealed only when the animals are metabolically challenged by eating a high fat diet. Various interventions can also markedly influence insulin sensitivity of adult and old rats. For example, consuming a high fat diet, reducing normal muscle contractile activity (eg, by hind-limb unloading, denervation, or immobilization), or various drugs that are often used to treat adult and older individuals can induce insulin resistance (2,26–29). Conversely, either calorie restriction or exercise can substantially improve insulin sensitivity in skeletal muscle of old rats (9–11,30–32). Our new approach makes it possible, for the first time, to determine the fiber type–specific influence of these various interventions on skeletal muscle from adult or old rats at the cellular level.

Although our primary focus in the current study was on glucose uptake, the results also demonstrated that this experimental approach has the potential value for understanding other issues related to aging in skeletal muscle. Sarcopenia refers to an age-related loss of muscle mass or muscle function. The extent of age-related muscle atrophy varies by fiber type, with type IIB fibers often found to be highly susceptible to loss of cross-sectional area. In the current study, there were no significant differences in the volume of any of the fiber types studied. Some skeletal muscles are characterized by age-related decrements in various mitochondrial markers, including enzyme activity and protein abundance (33). However, previous studies measuring mitochondrial enzymes in the whole epitrochlearis muscle reported no age-related changes in rats of ages similar to those in the current study (34–36). Consistent with these earlier results, we also found no significant difference of age in COXIV abundance in the whole epitrochlearis fiber bundle. However, there was a small, but significant main effect of age on COXIV abundance at the single-fiber level, with a greater COXIV levels in the fibers from 25-month-old rats. This result provides an example of the ability of single-fiber analysis to reveal differences that may be overlooked with whole tissue analysis.

Hybrid fibers have been reported to account for up to 5% to 30% of fibers of some rodent skeletal muscles (7). Because conventional tissue analysis cannot reveal information about the metabolic properties of hybrid fibers, single-fiber analysis is uniquely valuable to provide insights into the metabolism of hybrid fibers. In the current study, insulin-stimulated glucose uptake of IIB/X fibers did not differ significantly from type IIB fibers, whereas in our previous study of younger (2 to 3 months old) rats, we found that insulin-stimulated glucose uptake of type IIB/X fibers was slightly (25%) but significantly greater than for type IIB fibers. It is uncertain if the reason for the differing results from the current study compared with the earlier study is attributable to the difference in age of the rats. The IIB/X and IIB fibers in the current study were similar with regard to some characteristics (COXIV abundance and glucose uptake), but they differed with regard to others (APPL1 abundance and fiber volume). It would be useful to further characterize the protein expression profile of hybrid fibers from adult and old rats.

In conclusion, age-related changes in the structure and function of skeletal muscle do not occur uniformly across the different muscle fiber types. It is essential to develop and employ new experimental approaches to fully understand the effects of aging on skeletal muscle at the cellular and molecular level. In this context, the results of the current study provide a unique opportunity for future research focused on the metabolic properties of single fibers from adult and old rats. This new method will be valuable to test for possible age-dependent and fiber type–specific effects of various clinically relevant interventions (eg, dietary manipulations, exercise, limb immobilization, treatment with drugs, etc.) on glucose uptake, MHC isoform expression, fiber size, and protein abundance in adult and old rats.

Funding

This work was supported by the National Institutes of Health (AG-010026 to G.D.C.) and the American Heart Association (12PRE9270001 to J.G.M.).

References

1. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 2001. 86 3574–3578 [DOI] [PubMed] [Google Scholar]
2. Catalano KJ, Bergman RN, Ader M. Increased susceptibility to insulin resistance associated with abdominal obesity in aging rats. Obes Res 2005. 13 11–20 [DOI] [PubMed] [Google Scholar]
3. Nishimura H, Kuzuya H, Okamoto M, et al. Change of insulin action with aging in conscious rats determined by euglycemic clamp. Am J Physiol 1988. 254(1 Pt 1):E92–E98 [DOI] [PubMed] [Google Scholar]
4. Escrivá F, Gavete ML, Fermín Y, et al. Effect of age and moderate food restriction on insulin sensitivity in Wistar rats: role of adiposity. J Endocrinol 2007. 194 131–141 [DOI] [PubMed] [Google Scholar]
5. Sharma N, Arias EB, Sajan MP, et al. Insulin resistance for glucose uptake and Akt2 phosphorylation in the soleus, but not epitrochlearis, muscles of old vs. adult rats. J Appl Physiol 2010. 108 1631–1640 [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Mackrell JG, Cartee GD. A novel method to measure glucose uptake and myosin heavy chain isoform expression of single fibers from rat skeletal muscle. Diabetes 2012. 61 995–1003 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Bloemberg D, Quadrilatero J. Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis. PLoS ONE. 2012;7:e35273. doi: 10.1371/journal.pone.0035273. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Cartee GD, Bohn EE. Growth hormone reduces glucose transport but not GLUT-1 or GLUT-4 in adult and old rats. Am J Physiol 1995. 268(5 Pt 1):E902–E909 [DOI] [PubMed] [Google Scholar]
9. Cartee GD, Briggs-Tung C, Kietzke EW. Persistent effects of exercise on skeletal muscle glucose transport across the life-span of rats. J Appl Physiol 1993. 75 972–978 [DOI] [PubMed] [Google Scholar]
10. 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 1994. 266(5 Pt 2):R1443–R1447 [DOI] [PubMed] [Google Scholar]
11. Sharma N, Arias EB, Bhat AD, et al. Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats. Am J Physiol Endocrinol Metab 2011. 300 E966–E978 [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Allaf O, Goubel F, Marini JF. A curve-fitting procedure to explain changes in muscle force-velocity relationship induced by hyperactivity. J Biomech 2002. 35 797–802 [DOI] [PubMed] [Google Scholar]
13. Castorena CM, Mackrell JG, Bogan JS, Kanzaki M, Cartee GD. Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not. J Appl Physiol 2011. 111 1106–1117 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Wang C. Insulin-stimulated glucose uptake in rat diaphragm during postnatal development: lack of correlation with the number of insulin receptors and of intracellular glucose transporters. Proc Natl Acad Sci USA 1985. 82 3621–3625 [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Brooks SV, Faulkner JA. Skeletal muscle weakness in old age: underlying mechanisms. Med Sci Sports Exerc 1994. 26 432–439 [PubMed] [Google Scholar]
16. Höök P, Sriramoju V, Larsson L. Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans. Am J Physiol, Cell Physiol 2001. 280 C782–C788 [DOI] [PubMed] [Google Scholar]
17. González E, Delbono O. Recovery from fatigue in fast and slow single intact skeletal muscle fibers from aging mouse. Muscle Nerve 2001. 24 1219–1224 [DOI] [PubMed] [Google Scholar]
18. Thompson LV. Contractile properties and protein isoforms of single skeletal muscle fibers from 12- and 30-month-old Fischer 344 brown Norway F1 hybrid rats. Aging (Milano) 1999. 11 109–118 [PubMed] [Google Scholar]
19. Fisher JS, Brown M. Immobilization effects on contractile properties of aging rat skeletal muscle. Aging (Milano) 1998. 10 59–66 [DOI] [PubMed] [Google Scholar]
20. Groskreutz JJ, Thompson LV. Enzymatic alterations in single type IIB skeletal muscle fibers with inactivity and exercise in 12- and 30-month-old rats. Aging Clin Exp Res 2002. 14 347–353 [DOI] [PubMed] [Google Scholar]
21. Cao Z, Wanagat J, McKiernan SH, Aiken JM. Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res 2001. 29 4502–4508 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Larsson L, Li X, Frontera WR. Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells. Am J Physiol 1997. 272(2 Pt 1):C638–C649 [DOI] [PubMed] [Google Scholar]
23. Snow LM, McLoon LK, Thompson LV. Adult and developmental myosin heavy chain isoforms in soleus muscle of aging Fischer Brown Norway rat. Anat Rec A Discov Mol Cell Evol Biol 2005. 286 866–873 [DOI] [PubMed] [Google Scholar]
24. Kovanen V, Suominen H, Heikkinen E. Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle. Eur J Appl Physiol Occup Physiol 1984. 52 235–242 [DOI] [PubMed] [Google Scholar]
25. Delp MD, Duan C. Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle. J Appl Physiol 1996. 80 261–270 [DOI] [PubMed] [Google Scholar]
26. Stump CS, Tipton CM, Henriksen EJ. Muscle adaptations to hindlimb suspension in mature and old Fischer 344 rats. J Appl Physiol 1997. 82 1875–1881 [DOI] [PubMed] [Google Scholar]
27. Henriksen EJ, Rodnick KJ, Mondon CE, James DE, Holloszy JO. Effect of denervation or unweighting on GLUT-4 protein in rat soleus muscle. J Appl Physiol 1991. 70 2322–2327 [DOI] [PubMed] [Google Scholar]
28. van Raalte DH, Ouwens DM, Diamant M. Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options? Eur J Clin Invest 2009. 39 81–93 [DOI] [PubMed] [Google Scholar]
29. Flint OP, Noor MA, Hruz PW, et al. The role of protease inhibitors in the pathogenesis of HIV-associated lipodystrophy: cellular mechanisms and clinical implications. Toxicol Pathol 2009. 37 65–77 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Xiao Y, Sharma N, Arias EB, Castorena CM, Cartee GD. A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats. Age doi:10.1007/s11357-012-9383-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Sequea DA, Sharma N, Arias EB, Cartee GD. Calorie restriction enhances insulin-stimulated glucose uptake and Akt phosphorylation in both fast-Twitch and slow-twitch skeletal muscle of 24-month-old rats. J Gerontol A Biol Sci Med Sci doi:10.1093/gerona/gls085 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Dean DJ, Cartee GD. Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. J Gerontol A Biol Sci Med Sci 1996. 51 B208–B213 [DOI] [PubMed] [Google Scholar]
33. Cartee GD. Aging skeletal muscle: response to exercise. Exerc Sport Sci Rev 1994. 22 91–120 [PubMed] [Google Scholar]
34. Young JC, Chen M, Holloszy JO. Maintenance of the adaptation of skeletal muscle mitochondria to exercise in old rats. Med Sci Sports Exerc 1983. 15 243–246 [PubMed] [Google Scholar]
35. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem 2000. 275 3343–3347 [DOI] [PubMed] [Google Scholar]
36. Gulve EA, Rodnick KJ, Henriksen EJ, Holloszy JO. Effects of wheel running on glucose transporter (GLUT4) concentration in skeletal muscle of young adult and old rats. Mech Ageing Dev 1993. 67 187–200 [DOI] [PubMed] [Google Scholar]

[CIT0001] 1. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab 2001. 86 3574–3578 [DOI] [PubMed] [Google Scholar]

[CIT0002] 2. Catalano KJ, Bergman RN, Ader M. Increased susceptibility to insulin resistance associated with abdominal obesity in aging rats. Obes Res 2005. 13 11–20 [DOI] [PubMed] [Google Scholar]

[CIT0003] 3. Nishimura H, Kuzuya H, Okamoto M, et al. Change of insulin action with aging in conscious rats determined by euglycemic clamp. Am J Physiol 1988. 254(1 Pt 1):E92–E98 [DOI] [PubMed] [Google Scholar]

[CIT0004] 4. Escrivá F, Gavete ML, Fermín Y, et al. Effect of age and moderate food restriction on insulin sensitivity in Wistar rats: role of adiposity. J Endocrinol 2007. 194 131–141 [DOI] [PubMed] [Google Scholar]

[CIT0005] 5. Sharma N, Arias EB, Sajan MP, et al. Insulin resistance for glucose uptake and Akt2 phosphorylation in the soleus, but not epitrochlearis, muscles of old vs. adult rats. J Appl Physiol 2010. 108 1631–1640 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0006] 6. Mackrell JG, Cartee GD. A novel method to measure glucose uptake and myosin heavy chain isoform expression of single fibers from rat skeletal muscle. Diabetes 2012. 61 995–1003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0007] 7.Bloemberg D, Quadrilatero J. Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis. PLoS ONE. 2012;7:e35273. doi: 10.1371/journal.pone.0035273. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0008] 8. Cartee GD, Bohn EE. Growth hormone reduces glucose transport but not GLUT-1 or GLUT-4 in adult and old rats. Am J Physiol 1995. 268(5 Pt 1):E902–E909 [DOI] [PubMed] [Google Scholar]

[CIT0009] 9. Cartee GD, Briggs-Tung C, Kietzke EW. Persistent effects of exercise on skeletal muscle glucose transport across the life-span of rats. J Appl Physiol 1993. 75 972–978 [DOI] [PubMed] [Google Scholar]

[CIT0010] 10. 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 1994. 266(5 Pt 2):R1443–R1447 [DOI] [PubMed] [Google Scholar]

[CIT0011] 11. Sharma N, Arias EB, Bhat AD, et al. Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats. Am J Physiol Endocrinol Metab 2011. 300 E966–E978 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0012] 12. Allaf O, Goubel F, Marini JF. A curve-fitting procedure to explain changes in muscle force-velocity relationship induced by hyperactivity. J Biomech 2002. 35 797–802 [DOI] [PubMed] [Google Scholar]

[CIT0013] 13. Castorena CM, Mackrell JG, Bogan JS, Kanzaki M, Cartee GD. Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not. J Appl Physiol 2011. 111 1106–1117 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0014] 14. Wang C. Insulin-stimulated glucose uptake in rat diaphragm during postnatal development: lack of correlation with the number of insulin receptors and of intracellular glucose transporters. Proc Natl Acad Sci USA 1985. 82 3621–3625 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0015] 15. Brooks SV, Faulkner JA. Skeletal muscle weakness in old age: underlying mechanisms. Med Sci Sports Exerc 1994. 26 432–439 [PubMed] [Google Scholar]

[CIT0016] 16. Höök P, Sriramoju V, Larsson L. Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans. Am J Physiol, Cell Physiol 2001. 280 C782–C788 [DOI] [PubMed] [Google Scholar]

[CIT0017] 17. González E, Delbono O. Recovery from fatigue in fast and slow single intact skeletal muscle fibers from aging mouse. Muscle Nerve 2001. 24 1219–1224 [DOI] [PubMed] [Google Scholar]

[CIT0018] 18. Thompson LV. Contractile properties and protein isoforms of single skeletal muscle fibers from 12- and 30-month-old Fischer 344 brown Norway F1 hybrid rats. Aging (Milano) 1999. 11 109–118 [PubMed] [Google Scholar]

[CIT0019] 19. Fisher JS, Brown M. Immobilization effects on contractile properties of aging rat skeletal muscle. Aging (Milano) 1998. 10 59–66 [DOI] [PubMed] [Google Scholar]

[CIT0020] 20. Groskreutz JJ, Thompson LV. Enzymatic alterations in single type IIB skeletal muscle fibers with inactivity and exercise in 12- and 30-month-old rats. Aging Clin Exp Res 2002. 14 347–353 [DOI] [PubMed] [Google Scholar]

[CIT0021] 21. Cao Z, Wanagat J, McKiernan SH, Aiken JM. Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res 2001. 29 4502–4508 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0022] 22. Larsson L, Li X, Frontera WR. Effects of aging on shortening velocity and myosin isoform composition in single human skeletal muscle cells. Am J Physiol 1997. 272(2 Pt 1):C638–C649 [DOI] [PubMed] [Google Scholar]

[CIT0023] 23. Snow LM, McLoon LK, Thompson LV. Adult and developmental myosin heavy chain isoforms in soleus muscle of aging Fischer Brown Norway rat. Anat Rec A Discov Mol Cell Evol Biol 2005. 286 866–873 [DOI] [PubMed] [Google Scholar]

[CIT0024] 24. Kovanen V, Suominen H, Heikkinen E. Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle. Eur J Appl Physiol Occup Physiol 1984. 52 235–242 [DOI] [PubMed] [Google Scholar]

[CIT0025] 25. Delp MD, Duan C. Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle. J Appl Physiol 1996. 80 261–270 [DOI] [PubMed] [Google Scholar]

[CIT0026] 26. Stump CS, Tipton CM, Henriksen EJ. Muscle adaptations to hindlimb suspension in mature and old Fischer 344 rats. J Appl Physiol 1997. 82 1875–1881 [DOI] [PubMed] [Google Scholar]

[CIT0027] 27. Henriksen EJ, Rodnick KJ, Mondon CE, James DE, Holloszy JO. Effect of denervation or unweighting on GLUT-4 protein in rat soleus muscle. J Appl Physiol 1991. 70 2322–2327 [DOI] [PubMed] [Google Scholar]

[CIT0028] 28. van Raalte DH, Ouwens DM, Diamant M. Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options? Eur J Clin Invest 2009. 39 81–93 [DOI] [PubMed] [Google Scholar]

[CIT0029] 29. Flint OP, Noor MA, Hruz PW, et al. The role of protease inhibitors in the pathogenesis of HIV-associated lipodystrophy: cellular mechanisms and clinical implications. Toxicol Pathol 2009. 37 65–77 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0030] 30. Xiao Y, Sharma N, Arias EB, Castorena CM, Cartee GD. A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats. Age doi:10.1007/s11357-012-9383-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0031] 31. Sequea DA, Sharma N, Arias EB, Cartee GD. Calorie restriction enhances insulin-stimulated glucose uptake and Akt phosphorylation in both fast-Twitch and slow-twitch skeletal muscle of 24-month-old rats. J Gerontol A Biol Sci Med Sci doi:10.1093/gerona/gls085 [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0032] 32. Dean DJ, Cartee GD. Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. J Gerontol A Biol Sci Med Sci 1996. 51 B208–B213 [DOI] [PubMed] [Google Scholar]

[CIT0033] 33. Cartee GD. Aging skeletal muscle: response to exercise. Exerc Sport Sci Rev 1994. 22 91–120 [PubMed] [Google Scholar]

[CIT0034] 34. Young JC, Chen M, Holloszy JO. Maintenance of the adaptation of skeletal muscle mitochondria to exercise in old rats. Med Sci Sports Exerc 1983. 15 243–246 [PubMed] [Google Scholar]

[CIT0035] 35. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem 2000. 275 3343–3347 [DOI] [PubMed] [Google Scholar]

[CIT0036] 36. Gulve EA, Rodnick KJ, Henriksen EJ, Holloszy JO. Effects of wheel running on glucose transporter (GLUT4) concentration in skeletal muscle of young adult and old rats. Mech Ageing Dev 1993. 67 187–200 [DOI] [PubMed] [Google Scholar]

PERMALINK

Fiber Type–Specific Differences in Glucose Uptake by Single Fibers From Skeletal Muscles of 9- and 25-Month-Old Rats

James G MacKrell

Edward B Arias

Gregory D Cartee

Abstract