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Am J Physiol Endocrinol Metab 293: E908-E915, 2007. First published June 26, 2007; doi:10.1152/ajpendo.00634.2006
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A transcription-dependent mechanism, akin to that in adipose tissue, modulates lipoprotein lipase activity in rat heart

Gengshu Wu, Liyan Zhang, Jitendra Gupta, Gunilla Olivecrona, and Thomas Olivecrona

Department of Medical Biosciences, Physiological Chemistry, Umeå University, Umeå, Sweden

Submitted 22 November 2006 ; accepted in final form 14 June 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme lipoprotein lipase (LPL) releases fatty acids from lipoprotein triglycerides for use in cell metabolism. LPL activity is rapidly modulated in a tissue-specific manner. Recent studies have shown that in rat adipose tissue this occurs by a shift of extracellular LPL toward an inactive form catalyzed by an LPL-controlling protein whose expression changes in response to the nutritional state. To explore whether a similar mechanism operates in other tissues we injected actinomycin D to block transcription of the putative LPL controlling protein(s). When actinomycin was given to fed rats, heparin-releasable LPL activity increased by 160% in heart and by 150% in a skeletal muscle (soleus) in 6 h. Postheparin LPL activity in blood increased by about 200%. To assess the state of extracellular LPL we subjected the spontaneously released LPL in heart perfusates to chromatography on heparin-agarose, which separates the active and inactive forms of the lipase. The amount of lipase protein released remained relatively constant on changes in the nutritional state and/or blockade of transcription, but the distribution between the active and inactive forms changed. Less of the LPL protein was in the active form in perfusates from hearts from fed compared with fasted rats. When glucose was given to fasted rats the proportion of LPL protein in the active form decreased. Actinomycin D increased the proportion that was active, in accord with the hypothesis that the message for a rapidly turning over LPL-controlling protein was being removed.

muscle; heart perfusion; postheparin plasma; heparin; actinomycin D

THE ENZYME LIPOPROTEIN LIPASE (LPL) has a central role in metabolism of triglyceride-rich lipoproteins and, hence, in energy homeostasis. LPL hydrolyzes the triglycerides and thereby makes fatty acids available for energy production or storage. The activity of the enzyme can be rapidly modulated in a tissue-specific manner. This presumably steers the flow of lipoprotein fatty acids to different tissues as an adaptation to the physiological situation (36). During exercise and in the fasted state the main destination is heart and skeletal muscles. In the fed state and at rest, adipose tissue becomes a main destination.

Many studies have been carried out on the short-term modulation of LPL activity in adipose tissue and heart of rats. Early studies (28, 46) led to the still prevailing view that different regulatory mechanisms are at play in the two tissues. In heart there is little or no change of total tissue LPL activity with the nutritional state, but Borensztajn and Robinson (8) demonstrated that the heparin-releasable LPL activity increased severalfold on fasting. Since heparin does not rapidly penetrate the vascular endothelium, they suggested that the heparin-releasable LPL represented the enzyme at the endothelium. This has been designated as the "functional LPL", since it is this fraction that can act on lipoproteins in blood (45). It was concluded that modulation of LPL in heart entailed a shift of the enzyme to or from the endothelium, in contrast to adipose tissue, where regulation was exerted on the amount of the enzyme (28). This notion of two separate mechanisms was supported by a demonstration in mice that heart and adipose tissue LPL are under the control of separate genes (3). It is now clear, however, that modulation of LPL activity occurs at a posttranslational level both in adipose tissue (11, 13, 32) and in heart (2, 13, 37).

We (5) have found that in adipose tissue the modulation of LPL activity involves a shift of the enzyme from an active to an inactive form. This process appears to engage only the extracellular LPL (48). Neither LPL mass nor LPL activity changed significantly within the adipocytes. Furthermore, our studies indicated that the inactive form of the enzyme could not be recruited into the active form to any significant extent. An increase of the LPL activity appeared to require synthesis of a new enzyme protein that was processed into the active form, secreted from the adipocytes, and retained its catalytic activity (6). Following up on early observations that LPL activity in adipose tissue increases when fasted rats are given actinomycin D (which blocks transcription) (12, 44), Bergö et al. (6) showed that the decrease of adipose tissue LPL activity that occurs on food deprivation requires that a gene, separate from the lipase gene, is turned on. Candidates are the recently described angiopoietin-like proteins 3 and/or -4 (Angptl-3 and -4) (22). A number of studies on genetic deletion or overexpression of the Angptl proteins have led to the view that these are key regulators of triglyceride metabolism that function by modulating LPL activity (18). A recent study shows that Angptl-4 binds with high affinity to the active dimeric form of LPL and converts it to inactive monomers (47). Angptl-4 mRNA in rat adipose tissue was found to turn over rapidly, and changes in the Angptl-4 mRNA abundance were inversely correlated to LPL activity both during the fed-to-fasted and the fasted-to-fed transitions. This suggests that Angptl-4 is a fasting-induced controller of LPL in adipose tissue, acting extracellularly on the native conformation in an unusual fashion, like an unfolding molecular chaperone.

In the present study we explore whether a similar mechanism might be at play in the heart. Several earlier observations seemed to be in line with this. A consistent finding (1, 2, 4, 8, 20, 23, 24) has been that in (rat) heart, heparin-releasable LPL changes much more than total tissue LPL activity, indicating that it is mainly the extracellular LPL that is being modulated. Liu and Olivecrona (23) studied synthesis and transport of LPL in perfused guinea pig hearts by using pulse-chase methodology. They found no difference between hearts from fed or fasted guinea pigs in the incorporation of [35S]methionine into LPL, in degradation of LPL protein, or in release of labeled LPL protein to the perfusion medium. Yet the spontaneous release of LPL activity to the medium was about twofold higher with hearts from fasted guinea pigs, and the LPL activity released by a 2-min heparin flush was about threefold higher (24). This implies that the difference between the fed and fasted animals was not in the amount of lipase protein synthesized or in its turnover but must have been in the activity state of the enzyme at or close to the endothelium.

For the present study we used the same approach as previously (6) in adipose tissue, i.e., to block transcription of the putative LPL modulator by actinomycin in vivo. This had a fasting-like effect to increase heparin-releasable LPL activity both in intact animals and in perfused hearts. To explore whether there is a shift in the activity state of LPL molecules at or near the endothelium, we have used heart perfusion to study the ratios between active and inactive forms of LPL spontaneously released into the medium after different pretreatments of the rats.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats were from Møllegaard Breeding Center (Ejby, Denmark) or from local breeding using this strain. The rats were kept in a well ventilated, temperature- (21°C) and humidity-controlled (40–45%) room with free access to a standard laboratory chow (Laktamin, Stockholm, Sweden) and tap water. The light in the room was on between 6 AM and 6 PM. In experiments where the rats were to be fasted, food was withdrawn and a grid was placed in the bottom of the cages to prevent coprophagia. For rats that were fasted 8 h or less, food was withdrawn at 8 AM. For 16-h-fasted rats, food was withdrawn in the late afternoon and the experiments conducted between 8 and 10 AM the next morning. The rats were killed by decapitation. The animal ethics committee in Umeå approved the protocols for the experiments.

Materials. The substrate for assay of LPL activity was an emulsion, prepared by Fresenius-Kabi (Uppsala, Sweden), containing 100 mg of soybean triglycerides and 10 mg of egg yolk phospholipids/ml and a trace amount of 3H-labeled triolein. Intralipid 20% was from Fresenius-Kabi. Heparin was from Lövens (Malmö, Sweden). Cholesterol and triglycerides in plasma samples were measured using kits from Roche Diagnostics (Mannheim, Germany). Actinomycin D was from Sigma (St. Louis, MO). A stock solution was prepared in ethanol and diluted into saline (0.154 M NaCl) to a final concentration of 2 mg/ml for intraperitoneal injection. The dose was 2 mg/kg body wt. All other reagents were of the highest commercial grade possible.

Heart perfusion. The coronary vessels were perfused with heparin-containing medium by a modified Langendorff procedure, employing nonrecirculating retrograde perfusion through the aorta as described (43). After removal from the chest the heart was immersed in ice-cold saline, and the beat ceased immediately. The aorta was rapidly cannulated and residual blood removed by a flush of ice-cold saline. The heart was then mounted and the temperature of the medium adjusted so that the liquid flowing away from the heart was between 34 and 37°C. Under these conditions, a regular heartbeat was quickly restored. When this was obtained the experiment started. In some cases basal perfusate was collected on ice for ≤10 min for analysis of the spontaneous release of LPL. In other experiments the medium was changed to medium containing 5 IU heparin/ml. Perfusate was collected on ice for 60 s (6–7 ml) unless otherwise specified. Samples of the perfusate were either analyzed for LPL activity and/or mass within 2 h after collection or were frozen at –70°C for later measurement of LPL activity and mass.

Chromatography on heparin-agarose. In some cases, active and inactive forms of LPL in heart perfusates were separated by chromatography on heparin-agarose using a gradient of NaCl as described (5).

Assays. Tissue samples were weighed, cut into pieces, and frozen at –70°C. Later they were thawed and homogenized in a Tris-Cl buffer (pH 8.2) containing detergents and protease inhibitors as described (5). The homogenate was centrifuged for 15 min at 3,000 rpm, after which the supernatant or the intermediate phase (between the floating fat droplets and the pellet in the case of adipose tissue) was used for assay of LPL activity and mass.

LPL activity was measured as described (5). Before assay of plasma or liver homogenates, the samples were preincubated on ice with antiserum to rat hepatic lipase (31). Two microliters of tissue homogenate or ≤10 µl of plasma or perfusion medium (triplicate samples) was incubated for 60 min at 25°C with substrate in the presence of 10 µl of heat-inactivated serum from fasted rats (as source of apolipoprotein CII) and 6% BSA. The total volume was 200 µl. After termination of lipolysis by addition of organic solvents, the fatty acids were extracted and counted for radioactivity. Hepatic lipase in liver homogenates or in plasma was assayed using a sonicated emulsion of [3H]oleic acid-labeled triolein in gum arabic as described (31). The medium contained 1 M NaCl to suppress LPL activity. One milliunit lipase activity represents 1 nmol of fatty acids released per minute.

Statistical analysis. Data are given as means ± SE. There were at least five rats in each group unless otherwise specified. Statistical significance between groups was calculated by Student's t-test.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Blocking transcription in vivo increases heparin-releasable LPL in several tissues. The starting point for this study was that, in the fasted state, extracellular LPL activity in adipose tissue is suppressed by a transcription-dependent process (48). The major question asked was whether a similar mechanism operates in other tissues as well. For this, we first studied the effect of blocking transcription on the amount of LPL that is washed out from tissues (adipose, heart, a skeletal muscle, spleen, and kidney) by injection of heparin in vivo (Fig. 1). The rationale is that heparin releases LPL from the endothelial surface into blood. The decline of LPL activity after injection of heparin should therefore reflect the amount of functional LPL activity, i.e., the LPL that is accessible from blood. Studies with perfused hearts have shown that functional LPL is high in the fasted state but suppressed in the fed state. In accord with this, heparin injection washed out more LPL activity from hearts of fasted compared with fed rats (Fig. 1). In the fasted rats 41% of the LPL activity disappeared in 5 min and 46% in 60 min, whereas in the fed rats only 15% was washed out in 5 min and 24% in 60 min. When fed rats were given actinomycin 6 h before the injection of heparin, the amount of LPL released increased to 28% in 5 min and 38% in 60 min (Fig. 1). These data indicate that blocking transcription has a fasting-like effect on LPL in rat heart.


Figure 1
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  		Fig. 1. Effect of actinomycin D on the amount of lipoprotein lipase (LPL) washed out from tissues by heparin. The design of this experiment was to inject a single dose of heparin and study the decrease of tissue LPL activity with time after the injection. Three conditions were studied: {blacktriangleup}, fed rats given an ip injection of actinomycin; bullet, fed rats given saline; {circ}, 16-h-fasted rats given saline. Six hours later, heparin (200 IU/kg body wt) was injected iv. Groups of 5 rats from each of the 3 conditions were killed before and 5 or 60 min after the injection of heparin. LPL activity remaining in the tissue was determined as detailed in EXPERIMENTAL PROCEDURES. Data are means ± SE of 5 rats in each group. The effect of actinomycin is significant (P < 0.05), comparing fed rats given actinomycin to fed rats given saline both at 5 min and at 60 min for heart and for soleus muscle. For adipose tissue there was no significant effect of actinomycin on the high LPL activity characteristic of the fed state. The activity was significantly lower at all 3 times in the adipose tissue of the fasted rats compared with fed rats given actinomycin or saline.

 
A similar response was seen in the soleus muscle (Fig. 1). In fed rats only 8% of LPL activity was washed out from this tissue in 5 min and 12% in 60 min. When the rats were given actinomycin these figures increased to 20 and 28%, respectively, similar to the values in fasted rats, 19 and 30%, respectively. These data indicate that blocking transcription relieves a suppression of functional LPL activity in skeletal muscle as well as in the heart.

Consistent with a previous study (48), actinomycin had no effect on adipose tissue LPL activity in fed rats (Fig. 1). This is expected since in this tissue heparin-releasable LPL activity is high, and not suppressed, in the fed state. The activity was lower in adipose tissue of fasted rats. The washout of LPL activity was more gradual for adipose tissue than for heart. In spleen (Fig. 1) and kidney (data not shown), heparin caused no significant reduction of LPL activity, and actinomycin had no effect.

Postheparin LPL represents a composite of LPL released from many tissues. In a previous study with fasted rats (6) we found that pre- and postheparin LPL activities were markedly increased 6 h after actinomycin. Our interpretation was that this reflected the increase in adipose tissue LPL activity brought about by actinomycin. We have now repeated that experiment using fed rats (Table 1). Actinomycin increased the preheparin activity by ~60% and the postheparin activity by ~200% (P < 0.05 for both; Table 1). In contrast, actinomycin had no effect on the hepatic lipase activity in liver or in pre- and postheparin plasma (Table 1). In accord with earlier studies (9, 43), the postheparin LPL activity was higher in fed than in fasted rats, presumably reflecting the higher activity in adipose tissue in the fed state.


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  		Table 1. The effect of Act D on the amount of LPL and hepatic lipase released into plasma by heparin

 
Effect of blocking transcription in vivo on plasma lipid levels. For this, fasted rats were given actinomycin. As a result, LPL activity in adipose tissue more than doubled, and there were also small but significant increases of LPL activity in heart and in soleus muscle (Table 2). These increases of tissue LPL activity occurred without any significant changes in LPL mass. Plasma triglycerides decreased by 40% (P < 0.05). The values for plasma cholesterol increased by 12%, but this did not reach statistical significance. LPL mass in plasma, which is mostly inactive enzyme protein (27), did not change (Table 2).


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  		Table 2. Activation of the LPL system when transcription is blocked

 
Studies with perfused rat hearts. To more directly study the distribution of LPL in heart, we turned to the Langendorf perfusion system. Heparin, added to the perfusion medium, releases a peak of LPL activity that is commonly taken to represent the LPL present at or near the vascular endothelium (1, 2, 4, 8, 20, 23, 24). The heparin-releasable activity is high in hearts from fasted rats and suppressed in hearts from fed rats (4, 8, 20, 24, 34). When fed rats were given actinomycin 2 h before heparin, the heparin-releasable fraction (defined as the LPL activity released in 1 min) increased from 33 ± 4 to 57 ± 7 mU·min–1·g–1 (P < 0.05). This occurred without any significant change in the LPL activity remaining in the heart, which was 719 ± 57 mU/g in the controls and 707 ± 38 mU/g in the rats given actinomycin.

Although these results are in line with the hypothesis of a transcription-dependent mechanism to suppress LPL activity, a possible confounding factor was that the rats ate little or nothing after injection of actinomycin. Hence, the response that we ascribe to a block of transcription could in fact be a fasting/stress response. Therefore, we studied the effect of food deprivation on the heparin-releasable LPL activity. There was no change after 2 h (45.6 ± 4.4 compared with 53.4 ± 3.3 mU·min–1·g–1), but the activity then increased at 3 and 4 h (108 ± 14.4 and 92.9 ± 3.4 mU·min–1·g–1, respectively). These changes occurred without any significant change in residual LPL activity in heart (666 ± 43 mU·min–1·g–1 in the fed controls and 680 ± 41, 624 ± 23, and 566 ± 15 mU·min–1·g–1 at 2, 3, and 4 h, respectively). Hence, the response to actinomycin appeared to be somewhat more rapid than the response to food deprivation (compare results above; actinomycin caused a significant change already at 2 h). To further test whether the response to actinomycin depended upon the feeding pattern, we used an experimental design from Pedersen and Schotz (34). They found that when fasted rats were force-fed a glucose solution, heparin-releasable LPL in heart decreased to levels well below those seen in fed rats. Following this protocol, fasted rats were force-fed glucose and given actinomycin intraperitoneally. Table 3 shows the result of two such experiments. In both cases, actinomycin prevented the suppression of LPL activity. In the first experiment, glucose feeding reduced the heparin-releasable LPL activity by >70% in 80 min. Actinomycin, given 1 h before the glucose, fully prevented this decrease. In the second experiment, the total time between actinomycin and when the rats were killed was increased to 4 h. In this setting, actinomycin not only prevented the glucose-induced reduction of heparin-releasable LPL activity but actually increased this activity by 60% (P < 0.05). These data illustrate that the glucose-induced reduction of heparin-releasable LPL activity is a rapid process that requires transcription.


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  		Table 3. Suppression of heart heparin-releasable LPL activity requires transcription

 
Active and inactive forms of LPL in heart perfusates. Our previous studies on modulation of LPL activity in adipose tissue (48) indicated that a main process is a shift of extracellular LPL from active to inactive. Therefore, we questioned whether the same happens in heart. This cannot be adequately studied by analysis of the heparin-released material because the two forms of the lipase have different affinities for heparin (25). We speculated that the basal release of LPL into the perfusate could be used as a surrogate probe of the state of extracellular LPL. Figure 2 shows that the basal release of LPL activity is related to the heparin-induced release; higher heparin-induced release was associated with higher basal release. This is in accord with previous observations (24).


Figure 2
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  		Fig. 2. Correlation between spontaneous and heparin-induced release of LPL activity into the heart perfusate. In this plot, results from several separate experiments are combined: {blacksquare}, normal fed; {square}, deprived of food for between 6 and 24 h; {blacktriangledown}, deprived of food for 8 h, then tube-fed 3 ml of 60% glucose solution and killed between 80 and 160 min later; {blacklozenge}, deprived of food for 8 h, then tube-fed glucose and 2 h later tube-fed 1 ml of Intralipid 20%. The rats were killed 3 h later. Values are mU/ml perfusate and g heart. Linear regression analysis returns the following equation: spontaneous release = (1.42 ± 0.426) + (0.0688 ± 0.00850) x (heparin-induced release), P < 0.0001.

 
To analyze the state of LPL in the spontaneous outflow from the perfused hearts, we subjected the perfusate to chromatography on heparin-agarose. Both the active and inactive forms of the lipase bind to the column but can be separated by elution with a salt gradient (30). The first peak contains inactive LPL; the second peak is the active form. Figure 3 shows representative patterns for perfusates from six different conditions. Quantitative data on LPL activity and mass in the fractions are given in Table 4. The total amount of LPL protein released into the perfusate was similar in all cases (Table 4), but the distribution of the lipase protein between the inactive and the active forms differed. In fed rats (Fig. 3A) there were about equal amounts of the lipase protein in the two peaks, whereas in fasted rats two-thirds of the lipase protein was in the second peak (active lipase; Fig. 3C). When fasted rats were given glucose the pattern shifted so that two-thirds of the lipase protein was in the first peak (Fig. 3E). In all three physiological states, fed, fasted, and force-fed glucose, pretreatment with actinomycin caused a shift of lipase protein toward the active form (second peak in the chromatograms; Fig. 3, B, D, and E). For instance, in rats force-fed glucose ~60% of the lipase protein was in the first (inactive) peak, but when the rats were given actinomycin before the glucose the pattern shifted so that 70% of the lipase protein was in the second peak. These results demonstrate that the differences in perfusate LPL activity were due primarily to changes in the distribution of the lipase protein between the active and the inactive forms.


Figure 3
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  		Fig. 3. The nutritional state and administration of actinomycin D change the distribution between active and inactive forms of LPL protein spontaneously released into the medium from perfused rat hearts. Fasting was overnight, and the time between administration of actinomycin D and when the rats were killed was 4 h. The time between tube-feeding glucose (3 ml of 60% solution) and when the rats were killed was 3 h. After stabilization of the perfusion, medium was collected for 10 min. Seventy milliliters of the perfusate was applied on a heparin-agarose column (1 ml) and eluted with a salt gradient from 0.1 to 2.0 M NaCl in 20 mM Tris buffer, pH 7.2, containing 10% glycerol and 0.1% Triton X-100. For each condition, 3 hearts were analyzed. The figure shows representative profiles for 1 of the 3 rats. Mean values for the groups are given in Table 4.

 

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  		Table 4. LPL activities and mass for hearts perfused as in Fig. 3

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The study indicates that a transcription-dependent mechanism for modulation of LPL, similar to that demonstrated in rat adipose tissue (6), also operates in heart and skeletal muscles. The salient observation was that when fed rats were given an injection of actinomycin, heparin-releasable LPL activity in heart and in soleus muscle increased. This implies an LPL-suppressing protein. Our interpretation is that, after actinomycin, the transcript for this protein and therefore the protein itself declines, and the suppression of LPL activity is relieved. The LPL-suppressing protein appears to convert the lipase from its active form to an inactive form. In accord with this, most of the LPL protein released into the medium during perfusion of hearts from fasted rats was in the active form, whereas with hearts from fed rats most of the LPL protein was inactive.

It appears that the rapid changes of LPL activities that occur in heart and in adipose tissue during the fasted-to-fed or fed-to-fasted transitions engage only or primarily extracellular LPL molecules. This was shown directly for rat adipose tissue (48). Studies with perfused rat hearts have consistently shown that heparin-releasable LPL activity changes severalfold with the nutritional state, but total LPL activity changes much less or not at all (2, 4, 8, 37). An et al. (2) have reported that there is no change of LPL mass or activity in the cardiomyocytes. Hence, only a fraction of heart LPL is engaged in the change of activity, and this fraction includes the LPL molecules that are accessible to heparin. This supports the hypothesis that the change in activity engages primarily extracellular LPL also in heart.

A unifying picture that takes into account the combined evidence from studies on nutritional modulation of LPL activity in adipose tissue/adipocytes and heart/cardiomyocytes is that the cells produce LPL at relatively constant rates and release a mixture of active and inactive LPL molecules (19, 50). The enzyme then moves toward and probably recycles around the endothelium (16, 29). During these processes the active enzyme molecules either retain their conformation or refold to an inactive form. Physical studies (33) have shown that the LPL molecule is metastable. The active form is a homodimer. There is rapid subunit exchange between dimers, but the subunits are prone to refold into a catalytically inactive form (26). This was originally described as "a built-in ability to self-destruct" (33). Recent studies (47) indicate that, in adipose tissue, Angptl-4 functions as an LPL-suppressing protein that binds with high affinity to the active dimeric form of LPL and converts it into catalytically inactive monomers. It is, however, unlikely that Angptl-4 is the main LPL-suppressing protein in heart. Kersten (15a) has reported that Angptl-4 mRNA increases in heart on fasting, and preliminary experiments in this laboratory (Sukonina V, unpublished observations) have confirmed this. One would expect the LPL-suppressing protein in heart to decrease, not increase, on fasting, when heparin-releasable LPL activity increases. An interesting possibility is that, in heart and perhaps skeletal muscles, the conversion to inactive LPL is mediated by an extracellular protease (15, 49) rather than by one of the angiopoietin-like proteins.

Modulation of LPL activity is a rapid process. There are several reports that cardiac heparin-releasable LPL can change quickly. For instance, an increase was observed 1 h after induction of hypoinsulinemia by diazoxide in rats (40). In the present study the actinomycin-mediated increase of heart heparin-releasable LPL was detectable within 2 h. On the other hand, the modulation is not instantaneous. Pedersen and Schotz (34) found that when fasted rats were tube-fed glucose, there was a lag time of about 60 min before heart heparin-releasable LPL started to decrease. This time course is in line with the hypothesis of an LPL-suppressing protein that is itself controlled by rapid changes in gene expression. A corollary is that this protein and its mRNA turn over rapidly.

Our studies indicate that overall LPL activity is usually suppressed to some degree by the conformational switch mechanism. Total body heparin-releasable LPL activity increased by about 200% in the fed rats studied here and by nearly 400% in the fasted rats studied before (6). Our interpretation is that, in the fasted state, the activity is suppressed in adipose tissue; in the fed state the activity is suppressed in heart and skeletal muscles. In both cases, a transcription block results in relief of the suppression with consequent increase in total body heparin-releasable LPL activity. Consistent with this, plasma triglyceride levels decreased after actinomycin.

There are many reports on conditions or agents that change heparin-releasable LPL activity in heart [e.g., feeding/fasting (34), insulin (40), induction of a diabetic state (39), fat feeding (35), blocking triglyceride clearance with Triton WR-1339 or similar agents (40), lysophosphatidylcholine (39), beta-adrenergic stimulation (1), corticosteroids (35), increased cardiac workload (1), and infusion of a fat emulsion (42)], and there are studies on the signal substances and transduction pathways involved (41). We have not made any attempts to explore under what conditions the present mechanism is the major one. Most likely, there are also other mechanisms that modulate heart LPL activity. For instance, Pulinilkunnil et al. (38) have provided evidence that release of LPL from cardiomyocytes can be regulated by processes that involve actin cytoskeleton reorganization.

Our studies focused on the heart, but the results from the experiment where we studied heparin washout of LPL in vivo (Fig. 1) indicate that a similar mechanism operates in skeletal muscles as well. It is clear that heparin-releasable LPL activity can be rapidly modulated by activity/inactivity of the muscle (7, 14) and in response to changes in nutritional state (20), but there is no direct data that allow a conclusion on whether the changes primarily engage extracellular LPL also in muscle. Our data indicate that a transcription-dependent process is involved in the rapid response to feeding/fasting. Bey and Hamilton (7) and Hamilton et al. (14) have shown that the same is true for the rapid decrease of heparin-releasable LPL activity that occurs on immobilization. Possibly related to this are observations by Davies et al. (10). They found that a synthetic rexinoid suppressed LPL activity in heart and skeletal muscle of rats in vivo. The compound also suppressed heparin-releasable LPL activity in cultured myocytes that stably expressed human LPL. Following a lag time of 1–2 h after the compound was added, heparin-releasable LPL activity decreased precipitously to less than 10% of that present in control cells. The effect of the compound was transcription-dependent since coaddition of actinomycin completely blocked the decrease in heparin-releasable LPL activity. LPL mRNA remained unchanged, and it is of note that the suppression occurred with LPL expressed from a heterologous LPL construct. These observations support the hypothesis that skeletal muscle can express a potent LPL-suppressing protein, as observed for adipose tissue and for heart.

It is of interest that the conformational switch mechanism acts to suppress, not activate, LPL activity. The implication is that the factors that regulate LPL gene expression set this at a relatively high level to ensure that the tissue can produce enough LPL to meet peak demands. Between these peaks, suppression of LPL activity by the conformational switch mechanism ensures that the enzyme does not generate fatty acids in excess of what the tissue can handle. In line with this argument, it is of interest to note that tissue-specific overexpression of LPL causes lipotoxicity (21) and tissue-specific insulin resistance (17).


   ACKNOWLEDGMENTS
 
Present address for G. Wu: CIHR Group on the Molecular and Cell Biology of Lipids, Department of Biochemistry, 320 Heritage Medical Research Centre, University of Alberta, Edmonton, AB T6G 2S2 Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Olivecrona, Univ. of Umeå, Physiological Chemistry, Bldg. 6M, 3rd floor, SE-90187 Umeå, Sweden (e-mail: thomas.olivecrona{at}medbio.umu.se)

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
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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