- Top of page
We have examined the regulation of lipoprotein lipase (LPL) activity in skeletal muscle during physical inactivity in comparison to low-intensity contractile activity of ambulatory controls. From studies acutely preventing ambulatory activity of one or both the hindlimbs in rats, it was shown that ≈90–95 % of the heparin-releasable (HR) LPL activity normally present in rat muscle with ambulatory activity is lost, and thus dependent on local contractile activity. Similarly, ≈95 % of the differences in LPL activity between muscles of different fibre types was dependent on ambulatory activity. The robustness of the finding that physical inactivity significantly decreases muscle LPL activity was evident from confirmatory studies with different models of inactivity, in many rats and mice, both sexes, three muscle types and during both acute and chronic (11 days) treatment. Inactivity caused a local reduction of plasma [3H]triglyceride uptake into muscle and a decrease in high density lipoprotein cholesterol concentration. LPL mRNA was not differentially expressed between ambulatory controls and either the acutely or chronically inactive groups. Instead, the process involved a rapid loss of the HR-LPL protein mass (the portion of LPL largely associated with the vascular endothelium) by an actinomycin D-sensitive signalling mechanism (i.e. transcriptionally dependent process). Significant decreases of intracellular LPL protein content lagged behind the loss of HR-LPL protein. Treadmill walking raised LPL activity ≈8-fold (P < 0.01) within 4 h after inactivity. The striking sensitivity of muscle LPL to inactivity and low-intensity contractile activity may provide one piece of the puzzle for why inactivity is a risk factor for metabolic diseases and why even non-vigorous activity provides marked protection against disorders involving poor lipid metabolism.
One of the most important environmental changes influencing human physiology and disease in recent years has been the decline of physical activity that was a routine part of most people's lives. The typical person in hunter-gatherer and agrarian societies spent almost the entire day performing low-intensity muscular work or ambulatory activity. Epidemiological research over the past 50 years has conclusively and repeatedly demonstrated that inactivity is a major risk factor for death, primarily due to increased coronary heart disease (CHD) (Morris et al. 1953; Haskell et al. 1994). Other disorders involving poor skeletal muscle lipid metabolism are now on the rise (syndrome X, type II diabetes, obesity). Epidemiological research has often focused on the increased risk of disease in the most physically inactive people. Although somewhat controversial, the general consensus is that the dose-response relationship between activity and disease is steep, i.e. the greatest benefit would come from getting the least active to become moderately active (Haskell et al. 1994; Blair & Brodney, 1999; Kesaniemi et al. 2001). There has been a paradigm shift in the recommendations from consensus panels emphasizing the importance of avoiding the ill effects of physical inactivity by frequently incorporating moderate activity into the daily routine, and not just the more intense types of formalized endurance exercise training. Thus, there is a growing need to understand the underlying processes most sensitive to inactivity and low-intensity activity.
LPL has been studied heavily because this enzyme has a central role in several aspects of lipid metabolism (Olivecrona et al. 1997; Goldberg & Merkel, 2001). LPL also has a major influence on the partitioning of triglyceride-derived fatty acid uptake between different tissues, plasma cholesterol metabolism and the subsequent downstream intracellular effects related to lipid availability. Most dramatically, a partial reduction in LPL function because of a specific polymorphism was associated with a 5-fold increase in the odds ratio for death and greater CHD (Wittrup et al. 1999). Studies using transgenic overexpression (Levak-Frank et al. 1997), intramuscular LPL DNA injection (Schlaepfer & Eckel, 1999), acute administration of LPL antibodies (Goldberg et al. 1988), gene knockout technology (Levak-Frank et al. 1997) or LPL-altering drugs have all led to the conclusion that LPL is critical for the tissue-specific uptake of triglyceride-rich lipoproteins by non-hepatic tissues. In a study of a cohort of 730 patients, those with documented CHD had less post-heparin LPL activity compared with healthy controls (Henderson et al. 1999), and a smaller study observed that the clearance of chylomicron-like triglyceride emulsions was delayed (Martins et al. 1995). Thus, compelling arguments have recently been made that the physiological modulation of LPL activity may contribute to the aetiology or prevention of the metabolic disorders.
Owing to the wide swings in the local metabolic demands of skeletal muscle, understanding the biochemical plasticity in this tissue has been of high interest in medical research (Hamilton & Booth, 2000). Most of the emphasis on mechanisms regulating muscle LPL has concerned high-intensity exercise. Less is known about the contrast of inactivity to low-intensity contractions. If the underlying mechanisms regulating LPL happen to be dependent on intensity, then there would be much to be gained by insights about lower intensities that are more safe and feasible to perform, especially by the obese and/or elderly. For example, ageing reduces LPL activity (Bey et al. 2001; Hamilton et al. 2001) and the very sedentary elderly are encouraged to walk moderately (Frandin et al. 1991). Thus, we sought to understand why regulation of LPL activity in muscles may be different during inactivity compared with muscles contracting in the low-intensity range of the physical activity spectrum in either control animals or animals walking slowly on a treadmill after inactivity. We tested the hypothesis that the normally high LPL activity in muscle (especially in oxidative muscle) is significantly decreased by physical inactivity compared with ambulatory controls and that restoring ambulation in previously inactive animals would raise muscle LPL activity. Insights regarding the responsible mechanisms were obtained from measurements of LPL activity and protein mass in the functionally important heparin-releasable fraction and the larger intracellular precursor pool during physical inactivity or low-intensity activity, as well as determining whether such changes involve differential expression of LPL mRNA. The study design also allowed determination of whether local stimuli associated with contractile activity are responsible for muscle LPL regulation, and made a comparison of fibre types. Completing these aims could give a new perspective about the physiological processes for how LPL is regulated during an important cause for metabolic disorders, namely physical inactivity.
- Top of page
Compelling arguments by others have pointed towards an optimal balance of tissue-specific LPL activity as a molecular target for common lipid disorders, and high LPL in muscle has been suggested to be more related to favourable lipid metabolism than high adipose LPL. This study is relatively unique from many of the previous studies on muscle LPL because it has sought to understand the steps of muscle LPL regulation associated with physical inactivity and low-intensity contractions. These novel insights on this relationship may be of use because it is now clear that alterations of key molecular steps for muscle LPL regulation that are sensitive to inactivity can be prevented and reversed by even the non-fatiguing contractions that are relatively easy and safe to perform, and thus may provide an additional reason to support the public health message based largely upon epidemiology (Blair & Brodney, 1999; Kesaniemi et al. 2001). We found that almost all (up to 95 %) of the endothelial-enriched HR-LPL activity normally present in the capillaries of muscles was dependent upon low-intensity ambulatory activity (Fig. 1, Table 2). The conclusion that muscle LPL activity decreases during inactivity and is very sensitive to contractions was based upon a robust set of data. That is: (1) a rapid loss of LPL activity was replicated in several independent groups of rats; (2) LPL activity decreased in the three hindlimb muscle types examined; (3) LPL activity was rapidly reversed by slow treadmill walking; (4) the loss of LPL activity was observed in both sexes and also in mice; (5) changes in LPL activity were verified with different assays for total homogenates and heparin eluates; (6) LPL activity results were in agreement with independent measurements of TG-derived fatty acid uptake in vivo; and (7) two different methods of inactivity were used and similar results were obtained. The effect was not cumulative because there was no statistical difference in the magnitude of the decrease in muscle LPL activity after 1 or 11 days of reduced activity (Fig. 1). Thus, the study includes the most complete information available on the effect of inactivity on LPL.
The large difference in LPL activity between inactive and active muscles after a short period of time adds insights into why LPL is often reported to be different between fibre types and possibly the differences between inactive and active people. It has long been known that LPL activity is generally severalfold greater in the red oxidative muscle types than in white glycolytic muscles in ambulatory control rats (Hamilton et al. 1998; Ladu et al. 1991). However, now we can see (Fig. 2) that removing the normally high level of postural support by oxidative muscles was sufficient to abolish the difference of LPL activity between muscle fibre types. This suggests that the difference in LPL activity between fibre types is primarily due to the level of recruitment in normal daily activity rather than intrinsic factors such as cellular lineage or neurotrophic factors. About a 7-fold range in muscle LPL activity has been shown several times before between young people, and Lithell et al. (1981b) suggested that this was related to capillary density. However, now we know that such large differences in HR-LPL between people (Herd et al. 2001) and rat muscles (present study) are more transient than capillarization or morphological features. Close inspection of individual responses to acute moderate activity has also alluded to the potential for muscle LPL activity to rise and fall in some people by the same striking magnitude as we reported (Lithell et al. 1981a; Herd et al. 2001). Thus it is now clear that local changes in metabolism during even moderate contractions are the most important physiological stimulus for LPL regulation in skeletal muscle.
A balanced appraisal of the existing LPL research shows that many, but not all, of the studies provide evidence that LPL is responsive to altered physical activity. A thorough enough picture is emerging about muscle LPL regulation to learn physiological lessons from even the negative results. Take for example the seemingly straightforward question, does prolonged walking acutely raise skeletal muscle LPL activity? Even well performed studies could lead to potentially erroneous conclusions about the sensitivity of muscle LPL to moderate intensity activity incorporated as part of the daily routine. The acute treadmill walking data are an example. There was a dramatic increase in HR-LPL activity in the oxidative muscles (soleus and RQ) of animals during walking that had been previously sedentary (Fig. 1). In contrast, there was absolutely no trend for an increase in these two oxidative muscles when LPL was already high from normal cage activity (Fig. 1B and C, and Fig. 4 upper panel). Furthermore, this null result at 8 m min−1 was not because of a sub-threshold exercise intensity, because prior work (Hamilton et al. 1998) in the two oxidative muscle types showed that running at a seven times faster speed for 3-4 h day−1 also had no effect on oxidative muscles with already high LPL (Fig. 4). It is also apparent that a true assessment of the magnitude of the change in regulation of LPL activity and protein in the hours following moderate exercise or at the onset of inactivity requires careful analysis of the complex temporal patterns of muscle metabolism.
Figure 4. Summary of the dose-response relationship between physical activity/inactivity and muscle LPL
Upper panel, summary of oxidative muscle sections (deep RQ and soleus); lower panel, summary of more glycolytic muscle sections (superficial white vastus lateralis and RF). Results for each muscle type are normalized to ambulatory control values. Absolute values for controls are shown in Fig. 2. Treadmill walking was at 8 m min−1. The data on the effects of low-intensity physical activity are from the current results, and those on high-intensity running (56 m min−1 3.5 h day−1) are from published work (Hamilton et al. 1998). The two studies used the same strain and vendor of rats.
Download figure to PowerPoint
Figure 4 suggests that there is not a ‘minimal intensity threshold’ for the benefits on LPL regulation, but this could depend on the fibre type examined or the point at which ‘control activity’ is defined. Furthermore, fast twitch white muscle fibres have a greater recruitment threshold and are unlikely to be used much in normal ambulatory activities (Hennig & Lomo, 1985), and also generally have low LPL activity and protein unless there is exposure to intense exercise (Fig. 4).
There are several reasons to believe that the local LPL reductions in the oxidative hindlimb muscles were secondary to a reduction in the local energy demand, and that a more generalized hormonal signal was not sufficient to explain the results. Using a model of unilateral unloading, we found that only muscles of the unloaded hindlimb had a decrease in LPL activity (Table 2). In contrast, neither of the same oxidative muscles in the contralateral loaded hindlimb or other non-hindlimb oxidative muscles had a change in LPL activity (Table 2). Smol and colleagues (2001) made the important observation that adding denervation to a tenotomized soleus muscle did not produce an added effect on the loss of LPL activity. HU decreases the load to a muscle and also causes a local reduction of EMG activity during the first 24 h (Alford et al. 1987) or longer (Riley et al. 1990). A reduced energy demand by HU muscle is also evidenced by the rapid and sustained decrease in the local blood flow rate (McDonald et al. 1992; Colleran et al. 2000), especially evident in the oxidative muscles with frequent recruitment during normal postural and ambulatory activities (Hennig et al. 1985). The decrease in LPL in the hindlimb was larger in the oxidative slow and fast twitch red muscles compared with glycolytic fast twitch white muscles (Fig. 2), probably because the oxidative muscles had the greatest LPL and contractile activity to begin with in the ambulatory control conditions. In contrast to hindlimb muscles, LPL activity in the heart and diaphragm remained unchanged during HU (Fig. 2). This could be interpreted simply by the fact that the heart and diaphragm must continue working even during reduced weight-bearing activity, and thus local signals for TG-derived fatty acid utilization remained intact.
The large decrease in LPL in hindlimb muscles during inactivity was independent of a change in LPL mRNA concentration. LPL mRNA concentration remained unchanged in the soleus or RF muscles after both acute and prolonged intermittent (10 h day−1 for 11 days) inactivity (Table 3). The present data confirm and extend the recent finding with other methodologies that 12 h of inactivity in the soleus did not change LPL mRNA (Bey et al. 2003). We do not know whether more than 11 days would eventually decrease LPL mRNA significantly. However, even if this happened, the fact that the mRNA change would follow the more rapid decrease in LPL activity observed after just 1 day implies that the pretranslational mechanisms may not be causative, but merely associative or additive. Even though ageing is the most chronic condition causing physical inactivity, ageing decreases LPL activity and protein in the oxidative soleus muscle without any change in LPL mRNA concentration (Bey et al. 2001). As a caveat, we reported that unloading muscle (24 h day−1) by casting (Hamilton et al. 1998) or hindlimb suspension (present study) decreased LPL mRNA concentration. However, in the rat (but not human) profound muscle wasting takes place in 1 week of continuous unloading (Howard et al. 1989; Riley et al. 1990) that involves a large loss of both the muscle mass and total RNA (-56 %). Such effects were not occurring with prolonged intermittent inactivity. Also, because LPL activity and protein decreased at 12 h and the LPL mRNA was slower in decreasing at 7 days of continuous HU, it is not reasonable to suspect that the cause of the decrease in muscle LPL activity during continuous inactivity was a decrease in mRNA concentration. Instead we suggest that this decrease in LPL mRNA was a secondary consequence of further muscle adaptations induced by continuous inactivity, perhaps related to muscle wasting and remodelling.
The potential for modulating LPL mRNA concentration is apparently dependent on fibre type recruitment and/or exercise intensity. Relatively strenuous exercise can increase LPL mRNA concentration (Seip et al. 1997; Hamilton et al. 1998) locally in glycolytic fibre types (Hamilton et al. 1998). Lower intensity treadmill or voluntary walking is apparently sub-threshold for this exercise response, as in the present study. Simsolo et al. (1993) concluded that de-training of active people leads to a profound loss of skeletal muscle LPL activity, largely because of post-translational processes. Thus, the stable LPL mRNA concentration and ActD results during treadmill walking suggest that LPL transcription is not essential for up-regulating LPL activity at the low end of the dose-response curve. It remains to be seen whether LPL mRNA induction during intense contractions is essential for raising LPL activity.
The timing of the LPL protein mass changes also provides clues about the underlying process. Note that at the early time of 6 h, HR-LPL protein had decreased by the same percentage as HR-LPL activity, but the total LPL protein (primarily intracellular) had still not decreased significantly (Table 4). This may mean that there is a process causing a loss of LPL protein at the endothelium or other heparin binding sites before the larger precursor pool of LPL inside cells is drained.
Researchers interested in discovering treatments for lipid disorders have aggressively sought to discover the physiological and molecular factors that might be necessary to optimally maintain LPL activity, especially tissue-specific LPL patterns. Thus, it is impressive to observe that nothing else has a more deleterious effect on muscle HR-LPL than inactivity and to note the powerful counteractive influence of moderate amounts of contractile activity. The findings with ActD (Table 5) suggest a nuclear event is involved in the signalling for the loss of LPL in muscles. It has been known for a long time that ActD can raise LPL activity in adipose tissue (Schotz & Garfinkel, 1965; Wing & Robinson, 1968; Bergo et al. 2002). The lack of effect of ActD on active muscles and, instead, a preventative effect in the inactive muscle, provides the interesting evidence that the LPL inhibitor gene(s) is only expressed during inactivity or that physical inactivity ‘sensitizes’ muscle to it. In contrast, the same transcriptional inhibitor did not have a significant effect either on the maintenance of high skeletal muscle LPL activity in the ambulatory control animals (Table 5) or on the large rise in the LPL activity induced by treadmill walking (data in text of Results).
LPL has a central role in lipid metabolism and has been studied in literally hundreds of papers, including potential effects on the local uptake of plasma TG into the underlying muscle, local alterations in muscle glucose and fatty acid metabolism, alterations in HDL cholesterol, obesity and downstream factors related to atherosclerosis in animals that develop CHD. There is still much to be learned about the biochemical effects of changes in HR-LPL activity and protein during inactivity, but the most easily interpreted result with the HU model is the local decrease in [3H]TG uptake (Fig. 3). Since LPL activity and TG-derived fatty acid uptake in muscle are apparently related to energy demand, it follows that inactive muscle would have low lipid uptake in order to minimize unnecessary lipid accumulation and potential lipotoxicity.
Fasting plasma TG concentration is often not different between alternating days with prolonged walking or resting when the TG values are already low, even if muscle LPL activity is changing manyfold (Lithell et al. 1981a). Also, it is important to remember that the muscle mass of a rat hindlimb is only ≈6 % of body weight. Others concluded that LPL reductions during bed-rest may be in part related to a ≈20 % reduction of HDL-C, and a doubling of the average fasting VLDL TG concentration (Yanagibori et al. 1997, 1998). Raising energy demand with even moderate exercise can produce marked improvements in hypertriglyceridemic people, and produce consistent enhancement of normal subjects when challenged by a fat load (Frandin et al. 1991; Aldred et al. 1994; Herd et al. 2001).
A consensus panel concluded that altered TG metabolism exemplifies the strongest category of evidence for disease related to inactivity (Kesaniemi et al. 2001; Bouchard, 2001), in part because of "the strong and consistent effects of even one day of activity [on metabolism], well before traditional standards of ‘physical fitness’" (Thompson et al. 2001). It appears that muscle LPL regulation is one of the most sensitive metabolic responses to physical inactivity and low-intensity contractile activity. This may provide one piece of the puzzle in offering a plausible explanation for how inactivity is related to chronic diseases and why small amounts of activity provide marked protection against CHD (Kramsch, 1981). Because of the potential role of fatty acid-derived TG in signalling, it is possible that these changes in LPL and TG uptake are part of more widespread changes in muscle metabolism, as indicated recently by a change in gene expression for over 100 genes after several hours of inactivity (Bey et al. 2003).