INTRODUCTION

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LIPOPROTEIN LIPASE (LPL) is a key enzyme in fatty acid and lipoprotein metabolism in muscle (4, 18). LPL hydrolyzes triglycerides (TGs) associated with very-low-density lipoprotein (VLDL) and chylomicrons in circulating blood. The liberated fatty acids are ultimately stored in adipose tissue or oxidized in skeletal muscle. LPL also enhances VLDL receptor-mediated uptake of lipoprotein and indirectly plays a vital role in maintaining high-density-lipoprotein cholesterol (HDL-C) concentration (4). Thus low LPL activity is often associated with high plasma TG concentration, delayed and elevated postprandial lipids, and low HDL-C concentration (30, 31).

Aging is associated with several unhealthy metabolic changes. In addition to the well-known risk for increased coronary heart disease in humans, several studies have shown that plasma TG concentration increases with aging (7, 33; see Ref. 20 for review). A delayed clearance for chylomicrons and VLDL was reported in elderly subjects after an oral fat load (3, 10). Hypothetically, these changes in TG concentration and the delayed clearance of postprandial VLDL or chylomicrons from plasma with aging may partly involve a decrease of LPL activity. A 56-60% reduction of the post-heparin plasma LPL activity has been reported in the elderly compared with younger adults (6, 27).

The LPL protein is synthesized by parenchymal cells (adipocytes, cardiomyocytes, and skeletal muscle fibers). The secreted protein is transported in a dimeric form to the intimal surface of the vascular endothelium, where it will hydrolyze TGs in TG-rich lipoproteins (4, 18).

Several studies of LPL regulation during aging have focused on adipose tissue and heart (7, 8, 35). The consensus of these studies was a loss of LPL activity in these tissues with aging (7, 8). Skeletal muscle is a particularly important site of LPL regulation, because this tissue is ~45% of the body mass. It is the largest LPL-producing tissue and has a very high rate of TG extraction from plasma (26) and fatty acid oxidation (25). However, we are unaware of any studies on the effect of aging on LPL regulation in skeletal muscle. An understanding of the aging effects on LPL regulation is greatly needed (19).

The purpose of this study is to determine the effects of old age on skeletal muscle LPL. The regulation of LPL in skeletal muscle of young animals has often been shown to be different between muscle types. LPL mRNA concentration, LPL protein mass, and LPL activity were low in white glycolytic muscles and high in red oxidative skeletal muscles (20, 34). Moreover, different responses of LPL activity and protein have been observed between white glycolytic and red oxidative muscles with exercise and physical inactivity (20, 29). For this reason, the soleus muscle (a red oxidative postural muscle with predominately type I fibers) and the tibialis anterior (TA) muscle (a predominantly white glycolytic non-weight-bearing muscle with a majority of type IIb and IId/x fibers) were used to determine the effect of aging on LPL activity. LPL mRNA concentration and LPL protein mass were measured to begin understanding the process involved in LPL regulation during aging. Because of the potential decrease of post-heparin plasma LPL activity with aging (6, 27) and the fact that skeletal muscle has been shown to be a major site of LPL production, we hypothesized that LPL activity would also decrease in skeletal muscle with aging. Because skeletal muscle LPL, especially postural muscle, has been shown to be sensitive to reduced activity of daily living (ambulatory activity) (20), which increases during aging, we hypothesized that the decrease of LPL activity in skeletal muscle with aging would be muscle type dependent. We first measured LPL activity in 4- and 24-mo-old Fischer 344 (F-344) rats. Because those results indicated a statistical difference between the young (4 mo) and old (24 mo) adults, we then sought to extend our study to the F-344 × Brown-Norway strain after the first generation of breeding (F-344 × BN F1 rats) using middle-aged (18 mo) and old (31 mo) adults.

	METHODS

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Animal procedures. Two strains of rats with different life spans that are commonly used in aging research were utilized. Specific pathogen-free F-344 rats at 4 and 24 mo of age (n = 5) were used in our first comparison to study the effect of aging on LPL regulation in skeletal muscle. The choice of the 24-mo-old F-344 rat as the oldest is appropriate, because the mean life span was found to be 24 mo (36). F-344 × BN F1 rats, with a mean life span of 31 mo, were used as well (15).

Post-heparin eluate and tissue preparation. Heparin-releasable LPL (HR-LPL) was obtained from a nonfrozen soleus muscle mass (~50 mg) of each group of F-344 rats. This tissue was weighed, minced to 10-mg pieces, and soaked immediately in PBS containing 100 U/ml heparin (Sigma) for 30 min at 37°C. The eluate was stored at 80°C for subsequent measurements of HR-LPL activity.

LPL enzyme activity. Heparinized supernatants and heparin eluates were assayed for LPL enzyme activity using a [³H]triolein substrate as described by Nilsson-Ehle and Schotz (28). LPL activity was measured by the rate of hydrolysis of [³H]triolein containing substrate emulsified with lecithin in the presence of heat-inactivated rat serum as the source of apolipoprotein C-II and fatty acid-free albumin (28). Assays were performed at 37°C for 60 min. LPL activity was expressed as nanomoles of fatty acid hydrolyzed per gram per minute. Each sample was measured in duplicate and at two different concentrations to ensure reproducibility and linearity. The assay was specific to LPL, because lipase activity was not detectable when the sample was incubated with monoclonal anti-rat LPL antibody before addition of substrate.

Determination of LPL protein mass. LPL protein mass in the supernatants and post-heparin eluates was determined using a chicken polyclonal anti-bovine LPL antibody (gift from Dr. John Goers). Total protein (40-60 µg) from each sample was separated on 7% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride nitrocellulose membrane (Millipore). The membrane was blocked with 5% bovine serum albumin in buffer (25 mM Tris · HCl and 0.15 M NaCl, pH 7.4) for 1 h and then incubated with 1:5,000 polyclonal chicken anti-bovine LPL antibodies for 2 h. After incubation, the membrane was washed with a 0.05% Tween-Tris saline buffer and hybridized with 1:100,000 anti-chicken immunoglobulins conjugated with horseradish peroxidase for 1 h. The washes were repeated before the membrane was developed with the enhanced chemiluminescence (ECL+) kit (Amersham Pharmacia Biotech) and subjected to autoradiography. A 55-kDa band (Fig. 1) was detected as previously found in adipose tissue by Goers et al. (17). Rat heart LPL protein purified by affinity chromatography with a 1-ml heparin-Sepharose column (Amersham Pharmacia Biotech) and a purified bovine LPL protein (Sigma) were recognized at the same level of migration of LPL protein present in our muscle homogenate (Fig. 1, A and B). Membranes that were hybridized only with the secondary antibody did not reveal a nonspecific band near the LPL size (Fig. 1, B and C). The integrated optical density (IOD) of each band was quantified using a Personal Densitometer SI (Molecular Dynamics, Amersham Pharmacia Biotech) and ImageQuant (Molecular Dynamics).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Detection of lipoprotein lipase (LPL) protein in rat skeletal muscle. A: total protein sample from soleus (lane 1) was subjected to Western blotting using a chicken polyclonal antibody raised against bovine LPL. Total proteins from soleus homogenate were separated on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were hybridized with chicken polyclonal anti-bovine LPL antibodies and then with rabbit anti-chicken antibodies conjugated with horseradish peroxidase. LPL proteins were revealed by autoradiography. Rat heart LPL protein purified by heparin affinity chromatography (lane 2) and purified bovine LPL protein from Sigma (lane 3) were used as positive controls. Rat and bovine LPL proteins were detected similarly at ~55 kDa. B and C: membranes loaded with the same total protein samples from soleus (lanes 1 and 5), heart (lanes 2 and 6), heparin-releasable eluate from the soleus muscle (lanes 3 and 7), and purified rat LPL (lanes 4 and 8) were subjected to hybridization with chicken polyclonal anti-bovine LPL (B) or rabbit anti-chicken antibodies conjugated with horseradish peroxidase only (C). C: nonspecific bands with the secondary antibody.

Determination of LPL mRNA concentration. Northern blot analysis was performed with total RNA extracted from powdered muscle by the procedure described previously (20). Total RNA was isolated with the method developed by Chomczynski and Sacchi (12). Samples of total RNA were fractionated on a formaldehyde-agarose gel, transferred to nylon membranes, and hybridized for LPL transcripts with a random-primed ³²P-labeled rat LPL cDNA probe. The radiolabeled membrane was subjected to autoradiography with intensifying screens, and the IOD of each band was quantified. LPL mRNA concentration was expressed as LPL IOD per microgram of total RNA. The same membrane was subsequently hybridized with ³²P-labeled rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe. Values for LPL mRNA concentration were also normalized to GAPDH mRNA. There was no age effect on GAPDH IOD per microgram of total RNA. Dose-response analysis with increasing amounts of RNA was performed for each muscle group to verify that the IOD reflected quantitative changes in LPL mRNA concentration. Ethidium bromide staining was also performed to confirm the absence of RNA degradation and the equivalence of RNA loading.

Statistics. One-way analysis of variance was used to test the significance of aging on LPL activity, protein mass, and mRNA in skeletal muscles. When the analysis of variance was significant, a protected least significant difference Fisher's test was used for comparisons between ages. Results were considered significant at P < 0.05.

	RESULTS

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Effect of aging on total and HR-LPL activity in the soleus and TA muscles. HR-LPL was 52% (P < 0.05) less in the soleus muscle of 24-mo-old than 4-mo-old rats (Fig. 2A). Total LPL activity was also decreased 38% (P < 0.01) in the soleus muscle of 24-mo-old rats (Fig. 2B). Total LPL activity was fivefold greater (P < 0.01) in the soleus muscle than in the TA muscle of 4-mo-old F-344 rats (Fig. 2, B and C). However, total LPL activity in the TA muscle was not different between ages (Fig. 2C). The effect of aging on skeletal muscle LPL activity was confirmed in F-344 × BN F1 rats. Total LPL activity was decreased by 50% (P = 0.02) in the soleus muscle between 18 and 31 mo of age (Fig. 2D). Total LPL activity was fivefold greater in the soleus than in the TA muscle of 18-mo-old F-344 × BN F1 rats (Fig. 2, D and E). As with the F-344 strain, total LPL activity was not age dependent in the TA muscle of the F-344 × BN F1 rats (Fig. 2E).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Total and heparin-releasable LPL (HR-LPL) activities in skeletal muscle during aging. A: HR-LPL activity was measured in soleus muscle of 4- and 24-mo-old Fischer 344 rats. Minced soleus muscles (n = 5 in each group) were soaked in a 100 U/ml heparin-PBS for 30 min at 37°C. LPL activity was measured on each eluate. Total LPL activity was also measured in soleus (B) and tibialis anterior (C) muscles from 4- and 24-mo-old Fischer 344 rats (n = 5). Total LPL activity was determined in soleus (D) and tibialis anterior (E) muscles from 18- and 31-mo-old Fischer 344 × Brown-Norway F1 rats (n = 4). Values are means ± SE; each assay was run in duplicate. FA, fatty acid. *P < 0.05; P < 0.01 by one-way ANOVA.

Effect of aging on LPL mRNA concentration in the soleus and TA muscles. LPL mRNA concentration was measured in the soleus and TA muscles from 18- and 31-mo-old F-344 × BN F1 rats by Northern blot analysis (Fig. 3). LPL mRNA concentration (IOD/µg of total RNA or LPL/GAPDH) was not significantly different between ages in either muscle type (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   LPL mRNA concentration is not affected in skeletal muscle during aging. LPL mRNA concentration was measured in soleus (A and B) and tibialis anterior (C and D) muscles in 18- and 31-mo-old Fischer 344 × Brown-Norway F1 rats. Total RNA isolated from each skeletal muscle in each group (n = 5) was electophoresed, transferred to nylon membrane, and hybridized with radiolabeled rat LPL cDNA and then with rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA. mRNA was revealed by autoradiography. Integrated optical density (IOD) of LPL or GAPDH mRNA was quantified by densitometry. LPL mRNA concentration was expressed as IOD/µg total RNA (A and C). LPL mRNA concentration was also normalized with GAPDH mRNA concentration (B and D). Values are means ± SE. NS, not significantly different from 18-mo-old group by one-way ANOVA.

Effect of aging on total LPL protein mass in the soleus muscles. Total LPL protein mass was determined in the soleus muscle from 18- and 31-mo-old F-344 × BN F1 rats and 4- and 24-mo-old F-344 rats. In parallel with the decrease of total LPL activity in the soleus muscle between 18- and 31-mo-old F-344 × BN F1 rats, total LPL protein mass was decreased significantly by 46% (P < 0.05) with aging (Fig. 4A). Total LPL protein mass was also reduced significantly by 32% (P < 0.05) in the soleus muscle between 4- and 24-mo-old F-344 rats (Fig. 4B).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Regulation of total LPL protein in skeletal muscle during aging. LPL protein concentration was measured in soleus muscle in 18- and 31-mo-old Fischer 344 × Brown-Norway F1 rats (A) and 4- and 24-mo-old Fischer 344 rats (B) by Western blot using chicken polyclonal anti-bovine LPL antibodies. Total LPL protein concentration was quantified by densitometry. Values are means ± SE. *P < 0.05 by one-way ANOVA.

	DISCUSSION

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This study clearly demonstrates a large decrease in HR-LPL and total LPL activities in a red oxidative postural muscle (soleus) with aging in two different strains of rats. This decrease of LPL activity in skeletal muscle could potentially alter the use of TGs by muscle or contribute to other changes in lipid metabolism. We found that total LPL activity was 38% lower in the soleus muscle of F-344 rats at 24 mo of age than at 4 mo of age (Fig. 2B). Total LPL activity was also decreased by 50% in the soleus muscle between 18 and 31 mo of age in F-344 × BN F1 rats (Fig. 2D). A 52% decrease of HR-LPL activity was found in the soleus muscle between 4- and 24-mo-old F-344 rats (Fig. 2A). These results indicate a loss of LPL activity within muscle fibers and in the extracellular fraction associated with endothelial cells.

High concentration of TG in plasma is associated with an increased risk for coronary heart disease (14, 16). Overexpression of LPL in skeletal muscle has been shown to decrease plasma TG concentration, increase HDL-C level, and prevent diet-induced hyperlipidemia and obesity (23, 32). A 50% increase in the plasma TG concentration occurs between 6 and 27 mo of age in F-344 rats (2). This increase was associated previously with a low total LPL activity in adipose tissue in old rats (2). Now, we have also shown that the total and HR-LPL activity (present in the capillaries) within skeletal muscle is also significantly reduced during aging.

The LPL changes during aging were muscle type dependent. Total LPL activity and mRNA in the TA muscle, a white glycolytic non-weight-bearing muscle, was not affected by aging (Figs. 2 and 3). We reported in our study that total LPL activity was fivefold greater in the soleus than in the TA muscle for 18-mo-old F-344 × BN F1 rats and 4-mo-old F-344 rats. This difference of LPL activity between muscle types was attenuated with aging (Fig. 2). A muscle-type-dependent LPL regulation has been observed in several different muscles during exercise studies (20, 29). LPL activity was lower in white glycolytic than in red oxidative muscles of sedentary young adult rats (20, 29). Intense exercise training increased LPL activity in white glycolytic hindlimb muscles but did not induce an increase in the soleus muscle, in which LPL activity was already high in controls (1, 20).

However, the decrease of LPL activity and protein mass is unlikely to be caused by the change of fiber type in the soleus muscle during aging. Chen and Alway (11) reported a reduction in type IIa and IId/x fibers and a corresponding increase in the percentage of type I fibers in soleus muscles of aged F-344 × BN F1 rats. Thus the fiber type changes during aging are in the opposite direction to explain the observed decreases in LPL activity and protein in the soleus muscle.

The decrease of spontaneous cage activity associated with aging could potentially be responsible for the decrease in LPL activity with aging. Aging is associated with a reduction of physical activity in most species. Contractile activity is important to maintain LPL activity in muscle (20). LPL activity and protein fell significantly in the soleus muscle after 7 days of hindlimb immobilization compared with normal ambulatory activity (20), but it was unchanged in the TA muscle (unpublished observation; LPL activity: 98.7 ± 6.6 and 98.1 ± 7.8 nmol fatty acid · g¹ · min¹ in control and immobilized animals, respectively; LPL protein: 0.47 ± 0.03 and 0.45 ± 0.03 units/mg in control and immobilized animals, respectively). Thus, similar to aging, the effects of physical inactivity on muscle regulation are also muscle type dependent and predominantly in postural muscle. However, a caveat to this comparison is that LPL mRNA concentration in the soleus muscle of young rats decreased after complete immobilization (20).

Cartee (9) described other effects of aging on skeletal muscle lipid and carbohydrate metabolism and the impact of physical inactivity. Muscle capillarization and mitochondrial enzyme concentrations are reduced during aging in sedentary humans (13). In old rats, muscle capillarity is unchanged, whereas blood flow during contractile activity and muscle oxidative capacity are reduced. The effect of aging on oxidative capacity of muscle can be reversed by physical activity (24). After training, old rats and elderly humans attain a restoration of muscle oxidative capacity quite similar to that of their identically trained younger counterparts (9). More studies are needed to determine the impact of exercise on lipid metabolism in the elderly, but existing studies indicate that adaptation is preserved in the elderly (for review see Ref. 19).

This is the first study that has reported the effect of aging on LPL activity in a hindlimb skeletal muscle. Bergö et al. (5) found that soleus LPL activity was more responsive to feeding in 1-mo-old than in 9-mo-old Sprague-Dawley rats. Carlile and Lacko (7) reported an increase of LPL activity in epididymal adipose tissue between 2- and 24-mo-old Sprague-Dawley rats and a decrease of LPL activity in epididymal adipose tissue between 2- and 24-mo-old F-344 rats. A decrease of LPL activity has been found in the heart of F-344 rats with aging (7, 8) but not in Sprague-Dawley rats (7). Thus the effect of aging on LPL activity in adipose tissue and heart seems to be dependent on the rat strain. In contrast, we found a similar effect of aging on LPL activity and protein mass in the soleus and TA muscles of two different strains.

The underlying mechanism for the decrease in LPL activity in the soleus muscle with aging was apparently posttranscriptional. LPL mRNA concentration was not reduced in old rats (Fig. 3). Total LPL protein mass was decreased by 46% in the soleus muscle between 18- and 31-mo-old F-344 × BN F1 rats (Fig. 4A) and reduced by 32% between 4- and 24-mo-old F-344 rats (Fig. 4B). In contrast, Hotta et al. (22) found a decrease of LPL mRNA level in adipose tissue between young (7 yr) and old (30 yr) rhesus monkeys, suggesting that the mechanism of LPL regulation in adipose tissue with aging involves a pretranslational process. It is not uncommon that adipose tissue and skeletal muscle regulate LPL activity by different mechanisms.

In conclusion, we showed for the first time that total and HR-LPL activities and LPL protein mass are decreased in a red oxidative postural skeletal muscle with aging. These decreases were independent of a decrease of mRNA concentration, suggesting a posttranscriptional process with aging. However, this aging effect is dependent on the type of leg muscle, because it did not occur in a white glycolytic non-weight-bearing muscle. The mechanism of altered LPL regulation with aging is possibly different between the soleus muscle and the adipose tissue, because previous studies reported reduced LPL mRNA concentration in adipose tissue. The specific loss of LPL activity in an oxidative (soleus) muscle and not in the more glycolytic (TA) muscle during aging may be because contractile activity is a major determinant of muscle LPL activity (20) and because the low-intensity ambulatory activities that are reduced during aging would involve the soleus more than the TA muscle (21).