Aging is associated with elevated muscle triglyceride content and increased insulin-stimulated fatty acid uptake

Michelle Z. Tucker and Lorraine P. Turcotte

Department of Kinesiology and USC Diabetes Research Center, University of Southern California, Los Angeles, California 90089

Submitted 21 May 2002 ; accepted in final form 28 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 µU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased (P <= 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 72–145% in red and 112–129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration (r2 = 0.27, P < 0.05) and total TG synthesis rate (r2 = 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.

fatty acid oxidation; Fischer 344 x Brown Norway; glycogen; intramuscular triglycerides; insulin resistance; CD36; plasma membrane fatty acid-binding protein


ALTERATIONS IN SKELETAL MUSCLE fatty acid (FA) metabolism have been observed in connection with several metabolic disorders including insulin resistance (17) and obesity (16). Muscle triglyceride (TG) accumulation may be an important factor in the progression of these metabolic disorders. TG concentration was significantly elevated in muscle of obese subjects (22, 23) and in insulin-resistant muscle of obese Zucker rats (34). In addition, muscle TG concentration has been observed to be negatively correlated with insulin sensitivity (23, 31). Aging is associated with an accumulation of muscle TG as well as with an increased incidence of these metabolic disorders. In line with this, we (32) have shown that aging is associated with alterations in FA disposal under basal conditions. However, age-related changes in FA metabolism and substrate utilization have not been studied under insulin-stimulated conditions associated with high carbohydrate availability.

Muscle TG accumulation could be due to alterations in either FA uptake or disposal under basal or postprandial conditions. In obese subjects with normal glucose tolerance, muscle TG accumulation was attributed to a decrease in basal FA oxidation (16). Conversely, in type 2 diabetic subjects, postprandial FA metabolism may be more conducive to TG accumulation. Under postprandial conditions associated with hyperglycemia and hyperinsulinemia, FA uptake was increased and FA oxidation decreased compared with control subjects, whereas both FA uptake and oxidation were significantly decreased under basal conditions compared with normal controls (17). Thus alterations in either basal or insulin-stimulated FA utilization could favor TG accumulation. In old rats, we (32) have shown that basal FA oxidation is decreased. However, it is presently unclear whether FA uptake or disposal would be altered under postprandial or hyperglycemic and hyperinsulinemic conditions.

The metabolic factors responsible for the accumulation of TG with aging have not been determined. Studies on the effects of aging on FA metabolism are few and have focused on the basal state. However, in general, these studies have indicated that cellular FA disposal is altered with aging (7, 32). In humans, aging has been shown to be associated with a decrease in whole body FA oxidation at rest (7). We (32) have also observed a significant decrease in FA oxidation by hindlimb muscle of middle-aged and old rats compared with young rats under basal conditions. Considering that skeletal muscle can account for >50% of whole body FA disposal, it is critical to determine whether FA utilization pathways are similarly affected by age under hyperglycemic and hyperinsulinemic conditions.

Alterations in muscle FA disposal could be due in part to changes in the capacity of the muscle to take up FA from plasma. Several studies have found that alterations in FA uptake could be an important regulator of FA utilization in muscle via changes in FA transport capacity (4, 15, 35). We have shown (32) that, under basal conditions, FA uptake was not affected by age, but the effect of age on FA uptake under insulin-stimulated conditions remains to be determined. In addition, the relative partitioning of FA uptake toward oxidation or TG synthesis may be affected by advancing age. An additional consideration in the present study was the effect of glucose utilization on FA utilization. Because glucose uptake can affect FA uptake (36, 37), it is important to examine FA utilization under equivalent glucose uptake conditions. Insulin sensitivity has been found to either decrease (10) or stay the same (3) with advancing age, but evidence shows that insulin responsiveness remains unchanged throughout the aging process (18). Thus, to examine FA utilization under equivalent high glucose uptake conditions, a maximal hyperglycemic hyperinsulinemic protocol was utilized.

The purpose of this study was to determine whether FA metabolism under insulin-stimulated conditions is altered in muscle of old animals. This was accomplished by measuring palmitate uptake and disposal in the perfused hindlimbs of young, middle-aged, and old Fischer 344 x Brown Norway adult rats. FA disposal was assessed by the measurement of palmitate oxidation and incorporation into muscle TG.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. Male Fischer 344 x Brown Norway adult rats aged 5 (n = 8), 15 (n = 8), and 24 (n = 8) mo were obtained from National Institute on Aging (Bethesda, MD), housed singly, and maintained on a 12:12-h light-dark cycle. They received regular rat chow and water ad libitum. Ethical approval for the present study was obtained from the Institutional Animal Care and Use Committee at the University of Southern California.

Hindquarter perfusion. Animals were fasted overnight and anesthetized intraperitoneally with ketamine-xylazine (40 and 6 mg/kg body wt, respectively). A basal blood sample was taken via a tail vein. Then, the animals were prepared for hindquarter perfusion as previously described (25, 33). Before the perfusion catheters were inserted, heparin (150 IU) was administered into the inferior vena cava. The rats were killed with an intracardial injection of ketamine-xylazine immediately before the catheters were inserted, and the preparation was placed in a perfusion apparatus, essentially as described by Ruderman et al. (25).

The initial perfusate (300 ml) consisted of Krebs-Henseleit solution, 1- to 2-day-old washed bovine erythrocytes (hematocrit, 29%), 3.5% bovine serum albumin (Cohn fraction V; Sigma Chemical, St. Louis, MO), 20 mM glucose, 0.15 mM pyruvate, 1 mM albumin-bound palmitate, 1,000 µU/ml insulin, 8 µCi of albumin-bound [1-14C]palmitate (ICN Pharmaceuticals, Costa Mesa, CA), and 10 µCi of [3-3H]glucose (NEN Life Science Products, Boston, MA). The perfusate (37°C) was continuously gassed with a mixture of 95% O2-5% CO2, which yielded arterial pH values of 7.2–7.3 and arterial PCO2 and PO2 values that were typically 32–43 and 105–153 Torr, respectively, in all age groups. Mean perfusion pressures were 84 ± 9, 80 ± 12, and 67 ± 6 mmHg during unilateral hindquarter perfusion in the 5-, 15-, and 24-mo-old animals, respectively.

To minimize the possible effects of heparin added during surgery on plasma FA availability due to lipoprotein lipase activation, 25 ml of perfusate were passed through the circulatory system to remove remaining heparin and discarded. Subsequently, the perfusate was recirculated at a flow of 7 ml/min. Immediately after the beginning of the perfusion, the left superficial fast-twitch white (predominantly type IIb) and the deep fast-twitch red (predominantly type IIa) sections of the gastrocnemius muscles, as well as the plantaris muscle (mixed fiber types), were taken out and freeze-clamped with aluminum clamps precooled in liquid N2. The left iliac vessels were then tied off, and a clamp was fixed tightly around the proximal part of the leg to prevent bleeding. After an equilibration period of 20 min, the right leg was perfused at rest for 40 min at 7 ml/min (0.37 ± 0.01, 0.31 ± 0.01, and 0.33 ± 0.01 ml · min1 · g1 perfused muscle in 5-, 15-, and 24-mo-old rats, respectively). Arterial perfusate glucose concentration was held steady throughout the perfusion with a small volume-variable glucose infusion. Arterial and venous perfusate samples for the analysis of [14C]FA, 14CO2, and [3H]glucose radioactivities as well as for FA, glucose, and lactate concentrations were taken at 10, 20, 30, and 40 min. Arterial and venous perfusate samples for determinations of PCO2, PO2, and pH were taken at 10 and 30 min. Arterial samples for the determination of hemoglobin and hematocrit were taken 10 min before perfusion, and arterial perfusate samples were taken at 10 and 40 min for the determination of insulin. At the end of the 40-min perfusion period, muscle samples from the right leg of the animal were taken and treated as described above. The exact muscle mass perfused was determined by infusion of a black ink solution into the arterial catheter and weighing of the colored muscle mass at the end of the perfusions.

To correct for carbon loss, additional experiments were conducted to determine the acetate correction factor under our experimental conditions (27, 33). Thus, in subsamples of rats (n = 4 each for 5-, 15-, and 24-mo-old animals), hindquarters were perfused under identical perfusate conditions except that 5 µCi of [1-14C]acetate (ICN Pharmaceuticals) were added rather than [1-14C]palmitate and [3-3H]glucose. Arterial and venous perfusate samples were taken as described above and analyzed for [14C]acetate and 14CO2 radioactivities.

Blood sample analyses. Basal venous blood samples were analyzed for glucose, FA, and insulin concentrations. Arterial and venous perfusate samples were analyzed for glucose, lactate, and FA concentrations as well as for [14C]FA, 14CO2, and [3H]glucose radioactivities. Arterial perfusate samples were also analyzed for insulin concentration. Samples for glucose and lactate were put into 200 µM ethylene glycolbis({beta}-aminoethyl ether) (EGTA, pH 7) and immediately analyzed using the YSI SPORT lactate and glucose analyzers (Yellow Springs Instruments, Yellow Springs, OH). Samples for FA and insulin were put into 200 µM EGTA (pH 7) and centrifuged. The supernatant was collected and frozen until analyzed. FA concentration was determined spectrophotometrically by using the WAKO NEFA-C test (WAKO Chemicals, Richmond, VA), and insulin was determined by radio-immunoassay (Linco, St. Charles, MO).

To determine plasma palmitate radioactivity, duplicate 100-µl aliquots of the perfusate plasma were mixed with liquid scintillation fluid (BudgetSolve, Research Product International, Mount Prospect, IL) and counted in a Tri-carb liquid scintillation analyzer (model 2100TR; Packard, Meriden, CT) using a dual-tracer program. The liberation and collection of 14CO2 from the blood were performed within 2–3 min of anaerobic collection (2 ml) as previously described (33). Perfusate samples for the determination of PCO2, PO2, pH, and hemoglobin were collected anaerobically, placed on ice, and measured within 5 min of collection with an ABL-5 acid-base laboratory (Radiometer America, Westlake, OH) and spectrophotometrically (Sigma Chemical), respectively.

Muscle sample analyses. Muscle TG concentration was determined as glycerol residues after extraction and separation of the muscle samples, as previously described (29, 32, 33). To measure the incorporation of [14C]palmitate into muscle TG, lipids from the extracted organic layer were separated by liquid chromatography as previously described (33). To ensure that adipose tissue infiltration could not account for differences in muscle TG between groups, we measured muscle adipose-specific fatty acid-binding protein (aP2) protein content. In addition, FABPPM and CD36 protein contents were determined as indexes of FA transport protein content. FABPPM and aP2 protein contents were determined by Western blotting in plantaris muscle because it has a mixed fiber type composition that approximates that of the overall hindquarter preparation (1). Homogenates were prepared as described in detail (32). CD36 protein content was measured in red quadriceps muscle homogenate preparations from a subset of animals perfused under low glucose conditions (8 mM glucose, 1 mM palmitate, 25 µU/ml insulin) as previously described in detail (32). Solubilized muscle homogenate proteins, 100 µg for aP2 and CD36 and 50 µg for FABPPM, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked, rinsed, and then incubated with a purified polyclonal rabbit anti-aP2 (1:5,000, kindly donated by Dr. Judith Storch, Rutgers University, New Brunswick, NJ), purified monoclonal mouse anti-CD36 (1:2,000, Cascade Bioscience, Winchester, MA), or a purified polyclonal rabbit anti-FABPPM (1:3,000). For aP2, the secondary incubation was performed with goat anti-rabbit IgG (H+L)-alkaline phosphatase (1:2,500) followed by detection with enhanced chemiluminescence (ECL; Immun-Star; Bio-Rad) and exposure to film (Kodak XAR X-OMAT). Films were scanned using a Bio-Rad 710 Densitometer and quantitated using Quantity One software (Bio-Rad). An adipose tissue homogenate, prepared as previously described (26), was used as a 1-µg reference standard. For CD36 and FABPPM, the secondary incubation was performed with goat anti-rabbit IgG (H+L)-horseradish peroxidase (1:2,500) followed by detection with ECL (Super Signal West Pico; Pierce, Rockford, IL) and exposure to film (CL-Xposure, Pierce). Films were scanned using an HP ScanJet 6200C and quantitated using Scion Image (Scion, Frederick, MD). One hundred-microgram plantaris homogenate and 10-µg liver plasma membrane samples from young rats were used as reference standards for CD36 and FABPPM, respectively. For all target proteins, multiple gels were analyzed, and results were expressed as density units relative to the reference standard.

Muscle glycogen concentration was determined as glucose residues after hydrolysis of the muscle samples as previously described (9, 11, 32). To measure the incorporation of [3H]glucose into glycogen, an aliquot of the undiluted hydrolysate was mixed with liquid scintillation fluid (Research Product International, Mount Prospect, IL) and counted in a Tri-carb liquid scintillation counter.

Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and percent and total palmitate oxidation were calculated as previously described (32). Both percent and total palmitate oxidation were corrected for label fixation by using acetate correction factors of 1.311, 1.152, and 1.224 for 5-, 15-, and 24-mo-old animals, respectively. Oxygen and glucose uptake and lactate release were calculated by multiplying perfusate flow by the arteriovenous difference in concentration and were expressed per gram of perfused muscle, which was measured to be 5.5, 5.0, and 4.3% of body weight for unilateral hindquarter perfusion in 5-, 15-, and 24-mo-old animals, respectively. Muscle TG fractional synthesis rate was calculated as muscle TG-specific activity divided by arterial FA-specific activity. The rate of muscle TG synthesis was calculated as the product of muscle TG fractional synthesis rate and postperfusion muscle TG concentration (12). The glycogen synthesis rate was calculated as the 3H radioactivity recovered in glycogen divided by arterial glucose specific activity (24). For these calculations, the synthesis rates were weighted for fiber type composition (1). The calculation was derived with the assumption that type I and type IIa synthesis rates are approximately equal and are distinctly different from type IIb rates. Therefore, the glycogen synthesis rates for types IIa, IIb, and total hindlimb were calculated as

where SynW is white gastrocnemius (WG) synthesis rate, SynR is red gastrocnemius (RG) synthesis rate, IIa is type IIa synthesis rate, IIb is type IIb synthesis rate, and SynH is hindlimb synthesis rate.

The arterial and venous specific activities for palmitate and glucose did not vary over time and were not significantly different between groups. The arterial and venous specific activities averaged 35.3 ± 1.0 and 33.2 ± 1.0 µCi/mmol for palmitate and 1.9 ± 0.03 and 1.9 ± 0.03 µCi/mmol for glucose. Glucose concentration, glucose uptake, lactate release, and lactate concentration were also analyzed over the 40-min perfusion period to examine differences within each age group. However, because the calculated substrate utilization rates did not change significantly during the last 30 min of perfusion, the averages of the values were used to make comparisons between groups.

Statistical evaluation of the muscle TG and glycogen data was performed using a two-way ANOVA (Statistica, Tulsa, OK). Glucose concentration, glucose uptake, lactate release, and lactate concentration statistical analysis was done using an ANOVA with repeated measures. All other data were analyzed by a one-way ANOVA. Tukey's honestly significant difference test for post hoc multiple comparisons was performed when appropriate. Pearson product moment correlation coefficients were computed when applicable. In all instances, an {alpha} of 0.05 was used to determine significance.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Basal metabolic parameters. Basal venous blood glucose, plasma FA, and insulin concentrations were not significantly different between age groups (Table 1). Animal body weight increased with advancing age and was significantly higher in both the 15- and 24-mo-old animals compared with the 5-mo-old animals. Hindlimb muscle mass in the 15-mo-old animals was not different from that of the 24-mo-old animals but was 23% higher than that of the 5-mo-old animals.


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Table 1. Effects of age on basal metabolic parameters, body weight, and hindlimb muscle weight

 

Palmitate metabolism. As dictated by the protocol, perfusate palmitate concentration (974 ± 40, 1,091 ± 65, and 1,035 ± 44 µM for 5-, 15-, and 24-mo-old animals, respectively, P > 0.05) and palmitate delivery (264.8 ± 14.4, 232.5 ± 13.7, and 266.8 ± 14.3 nmol · min1 · g1 for 5-, 15-, and 24-mo-old animals, respectively, P > 0.05) did not vary over time and were not significantly different between age groups. Fractional palmitate uptake by 24-mo-old animals was 110% higher than by 5-mo-old animals and 44% higher than by 15-mo-old animals (Fig. 1A). This corresponded to total palmitate uptake rates that were 92 and 49% higher in 24-mo-old animals vs. 5- and 15-mo-old animals, respectively (Fig. 1B). The percentage of palmitate oxidized was 61% lower in the 15-mo-old animals compared with the 5-mo-old animals (Fig. 2A). Total palmitate oxidation by 24-mo-old animals was 88% higher than by 5-mo-old animals and 96% higher than by 15-mo-old animals (Fig. 2B).



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Fig. 1. Fractional (A) and total (B) palmitate uptake in perfused hindquarters of 5-, 15-, and 24-mo-old animals. Values are means ± SE for 5-mo-(n = 8, open bars), 15-mo-(n = 8, hatched bars), and 24-mo-old (n = 8, filled bars) animals. Because there were no significant changes in values measured after 20, 30, and 40 min of perfusion, average values were used for each animal. *P < 0.05 compared with 5-mo-old animals. +P < 0.05 compared with 15-mo-old animals.

 


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Fig. 2. Percent (A) and total (B) palmitate oxidation in perfused hindquarters of 5-, 15-, and 24-mo-old animals. Values are means ± SE for 5-mo-(n = 8, open bars), 15-mo-(n = 8, hatched bars), and 24-mo-old (n = 8, filled bars) animals. Because there were no significant change in values measured after 20, 30, and 40 min of perfusion, average values were used for each animal. Percent and total palmitate oxidation were corrected for label fixation as described in MATERIALS AND METHODS. *P < 0.05 compared with 5-mo-old animals. +P < 0.05 compared with 15-mo-old animals.

 

Substrate exchange across the hindquarter. Resting oxygen uptake did not vary over time and was not significantly different among the 5-, 15-, and 24-mo-old animals (38.7 ± 2.7, 36.1 ± 2.2, and 45.9.8 ± 3.2 µmol · g1 · h1, respectively, P > 0.05). As dictated by the protocol, arterial perfusate glucose concentrations did not vary over time and were not significantly different among the 5-, 15-, and 24-mo-old animals (20.0 ± 0.3, 20.0 ± 0.2, and 20.3 ± 0.5 mM for glucose, P > 0.05). Glucose uptake was not significantly different between age groups and did not change over time in 15-mo-(from 29.6 ± 2.9 to 36.8 ± 3.8 µmol · g1 · h1) and 24-mo-old (from 35.1 ± 3.3 to 43.2 ± 4.1 µmol · g1 · h1) animals. In the 5-mo-old animals, glucose uptake increased from 27.6 ± 4.6 at 10 min of perfusion to 45.4 ± 4.9 µmol · g1 · h1 at 40 min of perfusion (P < 0.05). Perfusate lactate concentration was not significantly different between age groups. However, arterial perfusate lactate concentration significantly increased over the perfusion period in the 5-mo-(from 1.7 ± 0.2 to 2.2 ± 0.2 mM, P < 0.05), 15-mo-(from 1.5 ± 0.31 to 2.0 ± 0.1 mM, P < 0.05), and 24-mo-old animals (from 1.5 ± 0.1 to 2.2 ± 0.1 mM, P < 0.05). Lactate release did not change significantly over time in any of the age groups but was significantly higher in 24-mo-old animals vs. 5- and 15-mo-old animals at all time points (P < 0.05), with average values of 9.2 ± 2.6, 9.8 ± 1.8, and 14.0 ± 1.0 µmol · g1 · h1 for the 5-, 15-, and 24-mo-old animals, respectively.

Muscle metabolite and protein content. Plantaris FABPPM protein content of 24-mo-old animals was 39% higher compared with 5-mo-old animals (P = 0.05) and 34% higher than in 15-mo-old animals (P < 0.10; Fig. 3). In addition, the concentration of FAT/CD36 protein was 57% higher in muscle of 24-mo-old animals compared with 5-mo-old animals (P < 0.05; Fig. 3). Plantaris aP2 protein levels were not significantly different between age groups, indicating that differences in muscle TG content between age groups were not due to adipose tissue infiltration. Expressed as a percentage of a 1 µg/µl adipose tissue homogenate standard, aP2 content was 7.1 ± 0.5, 8.1 ± 0.7, and 9.2 ± 0.7% for 5-, 15-, and 24-mo-old animals, respectively (P > 0.05; Fig. 4). Preperfusion RG and WG TG concentrations were 72–152% higher in 24-mo-old animals compared with 5- and 15-mo-old animals (Fig. 5A). Because there were no significant changes in muscle TG content during perfusion for all age groups, RG and WG remained significantly elevated (P < 0.05) in 24-mo-old animals compared with 5- and 15-mo-old animals at the end of the perfusion period. However, the rate of TG synthesis tended (P = 0.09 for both) to be increased in both RG and WG of 24- vs. 5- and 15-mo-old animals (Fig. 5B).



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Fig. 3. Fatty acid translocase (FAT/CD36) protein content in red quadriceps muscle of 5- and 24-mo-old animals (A) and plasma membrane fatty acid-binding protein (FABPPM) protein content in plantaris muscle of 5-, 15-, and 24-mo-old animals (B). Values are means ± SE for 5-mo-(open bars), 15-mo-(hatched bars), and 24-mo-old (solid bars) animals. CD36 (n = 6/group) and FABPPM (n = 8/group) protein contents were measured by immunoblotting muscle homogenates and quantitated by scanning densitometry. CD36 results are expressed as %plantaris muscle standard. FABPPM results are expressed as %liver plasma membrane standard. *P <= 0.05 compared with 5-mo-old animals. +P < 0.10 compared with 15-mo-old animals.

 


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Fig. 4. Adipose-specific fatty acid-binding protein (aP-2) protein content in plantaris muscle of 5-, 15-, and 24-mo-old animals. Values are means ± SE for 5-mo-(n = 8, open bars), 15-mo-(n = 8, hatched bars), and 24-mo-old (n = 8, solid bars) animals. aP2 protein contents were measured by immunoblotting muscle homogenates and quantitated by scanning densitometry. Results are expressed as %adipose tissue standard.

 


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Fig. 5. Muscle triglyceride (TG) concentration (A) and TG synthesis rate (B) in red and white gastrocnemius muscles of 5-, 15-, and 24-mo-old animals. Values are means ± SE for 5-mo-(n = 8, open bars), 15-mo-(n = 8, hatched bars), and 24-mo-old (n = 8, filled bars) animals. *P < 0.05 compared with 5-mo-old animals. +P < 0.05 compared with 15-mo-old animals.

 

The relationship between the rate of palmitate uptake and preperfusion muscle TG concentration in both RG (y = 14.7x0.43, r2 = 0.42, P < 0.05) and WG (y = 18.7x0.27, r2 = 0.16, P < 0.05) as well as total preperfusion TG concentration (y = 15.6x0.39, r2 = 0.27, P < 0.05, Fig. 6A) were exponential and found to be significant. In addition, the relationship between the rates of palmitate uptake and TG synthesis in both RG (y = 22.4x0.48, r2 = 0.63, P < 0.05) and WG (y = 37.0x0.54, r2 = 0.67, P < 0.05) as well as total TG synthesis rate (y = 30.6x0.54, r2 = 0.68, P < 0.05; Fig. 6B) were exponential and significant. Linear correlations between these variables were significant but slightly lower (r2 = 0.25 and 0.68 for palmitate uptake and total preperfusion TG concentration and total TG synthesis rate, respectively).



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Fig. 6. Correlation between palmitate uptake and total TG concentration (A) and total TG synthesis rate (B) in perfused hindquarters of 5-, 15-, and 24-mo-old animals. Total preperfusion TG content and total TG synthesis rate were calculated as described in MATERIALS AND METHODS. Regression equations and correlation coefficients are y = 15.6x0.39, r2 = 0.27, P < 0.05 (A) and y = 30.6x0.54, r2 = 0.68, P < 0.05 (B).

 

There were no significant differences in pre- or postperfusion glycogen concentrations in RG or WG muscles between age groups (Table 2). In addition, there were no significant age-associated differences in glycogen synthesis rates or change in glycogen content during the perfusion period in RG or WG muscles. However, in both muscles, the rate of glycogen synthesis tended (RG: P = 0.15, WG: P = 0.19) to be higher in 24- vs. 15-mo-old animals (Table 2).


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Table 2. Effects of age on muscle glycogen concentration and synthesis rates in red and white gastrocnemius muscles

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Our results show that aging is associated with alterations in muscle FA uptake and cellular disposal under insulin-stimulated conditions. In muscle perfused at equivalent high rates of glucose uptake, both FA uptake and FA oxidation were upregulated in muscle of the old animals. In addition, aging was associated with higher muscle protein expression of FAT/CD36 and FABPPM as well as higher preperfusion muscle TG levels in both red and white muscles. Both preperfusion muscle TG levels and TG synthesis rates were exponentially related with palmitate uptake. These results indicate that, under insulin-stimulated conditions, muscle of old animals demonstrates an increased ability to take up plasma FA, and this may favor TG accumulation in muscle.

With the use of the hindlimb perfusion system, plasma FA availability, blood flow, and capillary density are all factors that could have some impact on changes in muscle FA metabolism. In this experiment, blood flow and perfusate substrate concentrations were kept constant between groups, resulting in similar FA and glucose delivery to the perfused muscle of all age groups. Furthermore, because changes in fiber type distribution associated with aging have been shown to involve a small loss of type IIa fibers with a reciprocal increase in type I fibers (14), differences in muscle perfusion would be limited. A shift in muscle composition toward increasing intramuscular adiposity with a corresponding loss of muscle protein could also adversely affect FA metabolism. However, our data on the expression of the adipose tissue-specific protein aP2 in our muscle samples show that adipose tissue infiltration was minimal and not different between age groups in this study (26, 28). In addition, previous studies have indirectly indicated no significant differences in adipocyte infiltration into muscle in animals over the age range in our study (6, 14). These considerations suggest that, under the conditions imposed by our protocol, factors inherent to the perfused muscle mass must be predominantly responsible for the measured alterations in FA metabolism. Conversely, the use of the hindlimb perfusion system limits our overall conclusions regarding age-induced changes in FA metabolism because of the absence of an in vivo environment.

FA uptake was significantly increased in 24-mo-compared with 5- and 15-mo-old animals under insulin-stimulated conditions. Although these results are in contrast to the lack of difference in muscle FA uptake observed under basal conditions, they indicate that age-related alterations in FA uptake under insulin-stimulated conditions may be important to muscle TG accumulation (32). Over the past several years, data from several laboratories including our own have shown that FA uptake across the plasma membrane of muscle cells is dependent not only on the availability of FA transporter proteins but also on the ability to translocate these proteins to the plasma membrane. CD36 and FABPPM are two FA transporter proteins that have been linked to the regulation of FA uptake in muscle, and, in a manner reminiscent of GLUT4, CD36 and FABPPM have been shown to translocate to the plasma membrane of muscle cells with muscle contraction and with leptin or insulin treatment (5, 19, 20, 30). Thus the age-induced increase in FA uptake could be due to an increase in total transporter content and/or to an increase in the translocation of the transporters to the plasma membrane. In agreement with our previous findings, the present data show that FABPPM and FAT/CD36 content is elevated in muscle of old animals and that higher FA transporter content contributed to the observed increase in muscle FA uptake (32). The age-related increase in FA uptake may also have been associated with an increased translocation of CD36 to the plasma membrane with insulin stimulation.

Alterations in FA oxidation did not follow a consistent age-related pattern. In 15-mo-old animals, percent oxidation was significantly decreased by 38% compared with 5-mo-old animals. This occurred in combination with a slightly increased rate of FA uptake, which resulted in equivalent rates of total palmitate oxidation between 5- and 15-mo-old animals. In contrast, 5- and 24-mo-old animals oxidized similar percentages of palmitate. Because of the higher rates of palmitate uptake in the old animals, this resulted in significantly higher total rates of palmitate oxidation in the 24-mo-old animals. These results suggest that the partitioning of FA in favor of oxidation may be an attempt by the muscle of old animals to reduce the impact of high rates of FA uptake and limit accumulation of longchain acyl-CoA, lipid moieties whose presence has been linked to the development of insulin resistance (16, 21). Furthermore, our results suggest that the oxidative capacity of the muscle from old animals was sufficient to meet the demands imposed by our experimental conditions. Overall, aging was associated with an increase in the contribution of plasma FA to total aerobic energy production under insulin-stimulated conditions, increasing from ~10% in 5- and 15-mo-old animals to 15% in 24-mo-old animals.

As previously reported by us (32) and others (8), preperfusion muscle TG concentrations were significantly increased in red and white fibers of 24-mo-compared with 5- and 15-mo-old animals. Furthermore, there appears to be a gradual increase in TG accumulation with age, as preperfusion TG levels were increased by 43% in the red muscle of 15-mo-compared with 5-mo-old animals. Muscle TG accumulation with aging could be attributed to an increase in TG synthesis, a decrease in TG utilization, or both. Under insulin-stimulated conditions, there was a 50–84% increase in TG synthesis rate in the red (P = 0.09) and white (P = 0.08–0.09) gastrocnemius muscles of 24-mo-compared with 5- and 15-mo-old animals. This was related to the observed increase in FA uptake with age (r2 = 0.68, P < 0.05) and indicates that intracellular lipid availability may play a critical role in regulating FA disposal in muscle. Although the previously reported decrease in hormone-sensitive lipase protein content in muscle of old animals might suggest that TG utilization would decrease with aging, our estimates of the contribution of muscle TG to total aerobic energy production suggested that alterations in TG utilization under insulin-stimulated were minimal (32). Indeed, we estimate that TG utilization contributed 12–15% to energy production in all age groups. Thus our results suggest that higher rates of TG synthesis may play a more important role in the observed increase in muscle TG levels with aging.

As designed by our protocol, glucose uptake was not different between age groups. Because intracellular glucose disposal is affected by glucose uptake per se, we matched glucose uptake rates to eliminate differences in glucose uptake as a confounding variable. However, by using both maximal glucose and insulin stimulation, it is unclear whether glucose uptake was mediated by insulin or glucose. Although it is not known whether a single stimulus would have affected FA utilization differently, we believe that our results are reflective of equivalent intracellular glucose availability between age groups. As suggested by the higher rate of FA oxidation in the presence of similar oxygen consumption, glucose disposal should shift from oxidation toward increased glycogen storage in the 24-mo-old animals. In line with this, we estimated that plasma glucose contributed less to total aerobic energy production with aging, decreasing from 56–60% in the younger animals to 43% in the old animals. Although we did not find a significant increase in glycogen synthesis, our results show modest increases in glycogen synthesis rates (36–42%) in 24- vs. 5-mo-old animals and glycogen concentration accumulation (16–21%) over the perfusion period in the 24-mo-old animals. This is in line with results of others (2, 13), which suggest that the ability to synthesize glycogen is maintained in old animals.

In summary, the present study has shown that aging is associated with changes in cellular FA disposal in muscle perfused at equivalent high rates of glucose uptake. Aging was associated with increased rates of FA uptake and FA oxidation, and rates of FA uptake correlated with rates of TG synthesis. In addition, preperfusion TG levels and FABPPM and FAT/CD36 levels were elevated in muscle of old animals. These results suggest that the increase in FA uptake may be the initial factor in a cascade of FA disposal events that leads to the accumulation of muscle TG. Combined with the decrease in FA oxidation observed under basal conditions in old animals, the present results indicate that aging is associated with alterations in FA metabolism under both basal and insulin-stimulated conditions.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The present study was supported by grants from the American College of Sports Medicine and from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-45168).


    ACKNOWLEDGMENTS
 
We thank Hubert Chan, Nicholas Chan, Felicity Macahilig, and William Wu for expert technical assistance. We also thank Dr. Judith Storch for the generous donation of aP2 antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. P. Turcotte, Dept. of Kinesiology, Univ. of Southern California, 3560 Watt Wy, PED 107, Los Angeles, CA 90089-0652 (E-mail: turcotte{at}usc.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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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