The Journal of Nutrition Vol. 128 No. 6 June 1998, pp. 940-946

Mechanisms of Decreased Lipoprotein Lipase Activity in Adipocytes of Starved Rats Depend on Duration of Starvation^1,2

Jae-Joon Lee^{*, 3}, Pamela J. Smith^{, 4}, and Susan K. Fried^{*, 5}

^* Department of Nutritional Sciences, Cook College, Rutgers University, New Brunswick, NJ 08901-8525 and  Department of Medicine/Endocrinology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The aim of this study was to delineate the mechanisms by which varying periods of starvation decrease lipoprotein lipase (LPL) activity in rat adipose tissue. LPL mRNA levels and rates of LPL synthesis, degradation and secretion were compared in adipocytes from male rats that had been fed or starved for 1 or 3 d. The decreased LPL activity after 3 d of starvation (76%) was explained mainly by a 50% decrease in the relative abundance of LPL mRNA levels (P < 0.05) and a parallel 50% decrease in relative rates of LPL biosynthesis (P < 0.05). In contrast, starvation for 1 d decreased total LPL activity by 47% (P < 0.05) but did not affect LPL mRNA levels or relative rates of LPL biosynthesis. Pulse-chase studies demonstrated that 1 d of starvation increased the rate of degradation of newly synthesized LPL (P < 0.05) and markedly decreased its secretion into the medium (P < 0.05). A decrease in overall protein synthesis also contributed to the decreased LPL activity after 1 and 3 d of starvation. We conclude that the relative importance of pre- and post-translational mechanisms in regulating adipose tissue LPL activity depends on the duration of starvation. During short-term starvation, degradation of newly synthesized LPL is an important determinant to its secretion from the adipocyte and hence its functional activity at the capillary endothelium.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Lipoprotein lipase (LPL)⁶ catalyzes the hydrolysis of circulating triglycerides, allowing their utilization by peripheral tissues. In adipose tissue, LPL activity increases upon feeding and decreases with starvation. Thus LPL plays an important role in regulating rates of triglyceride deposition in adipocytes according to the energy needs of the organism. LPL regulation is quite complex because the enzyme is synthesized within the adipose cell and then secreted to its extracellular site of action, the luminal surface of the capillary endothelium (Braun and Severson 1992, Enerback and Gimble 1993). Despite much research attention, mechanisms of LPL regulation remain incompletely understood (Enerback and Gimble 1993).

The decrease in adipose tissue LPL activity during starvation in rats has been attributed to both pre- and post-translational mechanisms (Bergo et al. 1996, Doolittle et al. 1990, Ladu et al. 1991, Oliver and Rogers 1993). Discrepant results reported in the literature may have been due to the use of animals of different ages or to differences in experimental design and technique (Semb and Olivecrona 1989). Ladu et al. (1991) found a tight correlation between adipose tissue LPL activity and LPL mRNA levels in rats that were starved for 0, 1 and 6 d. In agreement with these results, Oliver and Rogers (1993) found that overnight starvation decreased relative rates of LPL synthesis by 50%, and Bergo et al. (1996) noted a 20-40% decrease in LPL mass in adipose tissue from rats starved overnight. However, Doolittle et al. (1990) used an experimental design in which adipose tissue from rats starved for 12 h was compared with that of refed controls (starved for 24 h and then refed with nonpurified diet supplemented with a 15% glucose solution for 12 h) to conclude that starvation increased adipose tissue LPL mRNA levels and rates of LPL synthesis. The decreased adipose tissue LPL activity in the starved rats was explained partially by a more rapid degradation of newly synthesized LPL and partially by a decreased LPL specific activity (Doolittle et al. 1990). It is not clear from that experiment whether the conclusions were limited to the comparison between a starved and a refed group. No previous studies have analyzed the turnover and secretion of LPL in adipocytes from starved vs. freely feeding rats.

Although LPL is an important determinant of the availability of exogenous fatty acids, rates of triglyceride synthesis are also dependent on the activities of a number of other steps in this pathway. In particular, the activation of fatty acids by acyl CoA synthetase (ACS) may also be regulated by nutritional and hormonal status (Shimomura et al. 1996, Weiner et al. 1992).

The primary goal of this study was to determine the effects of variable periods of starvation (1 or 3 d) on adipocyte LPL mRNA and rates of LPL biosynthesis, degradation and secretion. We also assessed the effect of starvation on the expression of acyl CoA synthetase (ACS) mRNA levels.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  All chemicals were obtained from Sigma Chemical, St. Louis, MO, unless otherwise noted.

Animals.  Male Wistar rats (4-5 wk of age) were purchased from Charles River Laboratories (Kingston, NY) and kept in our animal facility until they were 2-4 mo old as specified. The light cycle was from 0700 to 1900 h. Nonpurified diet (Ralston Purina, St. Louis, MO) and water were available at all times unless specifically noted. For starvation experiments, food was removed at 1600-1700 h, 1 or 3 d before the experiments, but water was provided. On the day of an experiment, at ~1100 h, rats were placed under light anesthesia in a CO₂-saturated chamber and killed by decapitation. Thus, the group starved for 1 d was deprived of food for 18 h. All procedures were approved by the Rutgers University Animal Care Committee.

Preparation of adipocytes and analysis of mRNA levels.  Preliminary data showed no differences in LPL activity between the epididymal and retroperitoneal adipose tissue; thus these two depots were pooled for subsequent analysis unless otherwise noted. Tissues from rats in fed and starved groups were processed in parallel. In experiments in which RNA was extracted from tissue and isolated cells, fat depots from two rats per group were pooled. Otherwise, tissue from individual rats was used. Adipose tissue was minced into small (30-50 mg) fragments. An aliquot (1 g) was placed in a 50-mL polypropylene test tube, immediately frozen in a dry-ice/methanol bath, and stored at 80°C until further analysis. Aliquots of tissue were also taken for determination of adipocyte number and tissue lipid content (DiGirolamo et al. 1971). The remaining tissue was digested for 1 h with collagenase (1 g/L, Worthington Biochemicals, Freehold, NJ) in Krebs Henseleit Buffer (KHB)(Wardzala and Cushman 1978), containing 30 mmol/L HEPES, 5 mmol/L glucose, 100 nmol/L phenylisopropyladenosine (PIA) and 40g/L bovine serum albumin (BSA, CRG7, Intergen, Purchase, NY). The digest was filtered through a 250-µm nylon mesh and the fat cells were allowed to float. The infranatant was removed and centrifuged at 1000 × g for 5 min to pellet the stromal-vascular cells. The fat cell and stromal fractions were then washed three times in KHB with 10 g/L BSA, and resuspended in KHB with 4 g/L BSA. After removing the wash medium, aliquots (~5 × 10⁶ cells) were immediately homogenized in guanidinium isothiocyanate buffer for RNA extraction (Chomczynski and Sacchi 1987). Northern blotting and slot blotting were conducted as previously described (Fried and Zechner 1989, Fried et al. 1992) utilizing cDNA probes for human LPL, mouse ACS (Weiner et al. 1992) and 28S ribosomal RNA.

Analysis of LPL biosynthesis.  These experiments were conducted essentially as previously described (Fried et al. 1992 and 1993). Aliquots of the adipocytes were resuspended in minimal essential medium (MEM) lacking methionine and cysteine but containing 4 g/L BSA, 100 nmol/L PIA and 5 mmol/L glucose. Fat cells were preincubated at 37°C for 30 min in the presence of 3.5 nmol/L insulin. At the end of this period, 3.7 GBq/L of Expre³⁵S³⁵S mix (New England Nuclear, Boston, MA) was added, and the incubation was continued for 15 or 30 min, as specified in individual experiments. Because the results of the starved vs. fed comparison did not depend on the labeling period, and incorporation of label into LPL was linear for 30 min (Lee, J.-J., Smith, P. J. and Fried, S. K., unpublished observation), the data using both pulse times were pooled for statistical analysis of the effects of starvation. At the end of the pulse-labeling period, adipocytes were concentrated and separated from the medium by centrifugation through 0.4 mL of silicone oil and immediately placed in lysis buffer containing proteolytic inhibitors at 4°C (Doolittle et al. 1990, Fried et al. 1992).

Pulse-chase experiments.  Cells were pulse-labeled for 15 min as described above except that in one of four experiments, no insulin was present. Data from all four experiments were pooled because the relative difference in LPL half-life in fed vs. starved groups was similar in experiments conducted with or without insulin present during the incubation. For chase incubations, after pulse-labeling, cells were washed four to five times with 20 mL of MEM, 40 g/L BSA, 100 nmol/L PIA containing 1.2 g/L methionine and 1.2 g/L cysteine (but no insulin). After resuspension in the same buffer at 10% v/v, cells were incubated at 37°C for up to 3 h. Aliquots of the cells were separated from the medium and homogenized in lysis buffer at specified times. The pulse and chase media were also saved at 80°C for immunoprecipitation (see below). Cell homogenates were sonicated, centrifuged at 14,000 × g (Eppendorf microfuge, Brinkman Instruments, Westbury, NY), and the infranatant below the fat cake and silicone oil was saved at 80°C for immunoprecipitation. Leakage of the cytosolic enzyme lactate dehydrogenase activity into the medium during adipocyte incubations was minimal (<5% of cellular values) in adipocytes of fed and starved rats, indicating that the appearance of newly synthesized LPL in the medium appeared to represent secretion (Fried et al. 1990).

Immunoprecipitation.  After precipitation with 12% trichloroacetic acid (TCA) to determine incorporation of ³⁵S label into total proteins, aliquots of cell homogenates from fed and starved rats containing equal counts (0.3-1 ×10⁶ ) were used for immunoprecipitation. LPL released from the fat cells into the medium (0.5- to 0.7-mL aliquots) were immunoprecipitated after the addition of 1% Triton-X-100. Immunoprecipitation was conducted as described by Doolittle et al. (1990) with the use of affinity-purified chicken anti-bovine LPL antibodies (3-5 µg, courtesy of J. Goers, California Polytechnic State University, San Luis Obispo, CA ), except that affinity purified goat anti-chicken immunoglobulin G (IgG) (Kirkagaard and Perry, Gaithersberg, MD) and Omnisorb or recombinant protein G (Calbiochem, La Jolla, CA) was used to precipitate immune complexes. Immunoprecipitated proteins were separated by SDS- PAGE (9%) and detected by fluorography. Scanning laser densitometry of fluorographs was used to quantitate the alterations in LPL biosynthesis and turnover. To quantitate relative rates of LPL synthesis (as a percentage of total protein synthesis), the LPL band or the corresponding location in a control immunoprecipitation (nonimmune chicken IgG) was cut out of the dried gels, digested in 30% H₂O₂ (v/v) for 24 h at 60°C and radioactivity was determined by scintillation counting. Total protein synthesis per gram of cell lipid or per 10⁶ cells was calculated. To calculate the rate of LPL release into the medium, the volume of medium used for immunoprecipitation was related to the cell number (or lipid weight) of adipocytes in the cell homogenate used for immunoprecipitation.

Measurement of LPL activity.  LPL activity releasable by heparin from tissue fragments and total LPL activity extractable from homogenates containing 5 g/L deoxycholate were measured as previously described (Fried et al. 1990, Iverius and Brunzell 1985). One unit of LPL activity is defined as the release of 1 µmol free fatty acid in 1 h.

Determination of fat cell size and number.  The number of cells per milliliter fat cell suspension was determined by dividing the lipid content (DiGirolamo et al. 1971) by the average fat cell weight determined after osmium fixation of adipose tissue fragments as described by Hirsch and Gallian (1968).

Analysis of data.  All statistical analyses were performed using SYSTAT software (version 3, Evanston, IL). Data are presented as means ± SEM. When appropriate, statistical significance was assessed using ANOVA with duration of starvation as a grouping factor, and when appropriate, insulin as a repeated measure. Post-hoc comparisons of group means were undertaken by Tukey's test when main effects were significant (P < 0.05) (Tukey 1977). To control for variability resulting from differences in the specific activity of ³⁵S-amino acids between experimental days, paired t tests were used to compare rates of protein synthesis in fed and starved rat adipocytes incubated in parallel. Other group comparisons were carried out by independent t tests.

	RESULTS

Abstract Introduction Methods Results Discussion References

Animals and LPL activity.  Rats (initially weighing 420 g, 4 mo old) lost an average of 30 g after 1 d and 58 g after 3 d of starvation. As shown in Table 1, starvation for 1 d decreased total extractable LPL in adipose tissue to 36% and heparin-releasable (HR) LPL activity to 19% of fed levels. Starvation for 3 d decreased total extractable (TE) LPL activity further, to 24% of control levels, and HR LPL activity fell to 15% of control levels. Fat cell size was not affected by 1 d of starvation (0.48 ± 0.06 in fed controls vs. 0.36 ± 0.04 µg lipid/cell after 1 d of starvation), but it was decreased by 3 d of starvation (0.33 ± 0.02 µg lipid/cell; P < 0.05 vs. fed, n = 6, independent t test).

LPL and ACS mRNA levels.  Northern analysis of adipocyte RNA showed that LPL mRNA levels were consistently decreased after 3 d but not 1 d of starvation (Fig. 1). To quantitate the changes in mRNA levels, slot blots were loaded with varying quantities of total RNA extracted from either total adipose tissue or isolated adipocytes and probed for LPL and ACS mRNA and 28S ribosomal RNA (rRNA) levels (Table 2). After 1 d of starvation, adipose tissue LPL mRNA (relative to rRNA) was not signficantly decreased compared with fed controls. After 3 d of starvation, LPL mRNA relative to rRNA ratios decreased by 57% compared with control-fed levels. Results were nearly identical to those obtained with the use of extracts of isolated adipocyte RNA, indicating that the LPL mRNA levels in adipose tissue reflect alterations in adipocyte expression. There was a trend (P = 0.1 by ANOVA) toward a decline in ACS mRNA with starvation (Fig. 1 and Table 2).

View larger version (45K): [in this window] [in a new window]   Fig 1. Northern analysis of lipoprotein lipase (LPL) and acyl CoA synthetase (ACS) mRNA expression in isolated adipocyte RNA from fed rats and rats starved for 1 and 3 d. Isolated adipocytes were prepared from combined epididymal and retroperitoneal fat depots of 4-mo-old rats that had had free access to food (fed), or had been starved for 1 or 3 d. After RNA extraction and electrophoresis, blots were hybridized with cDNA for hLPL, mACS and 28S ribosomal RNA as a loading control. Each lane represents adipocyte RNA from an individual rat.

In addition to adipocytes, adipose tissue contains other cell types, including preadipocytes. Therefore, LPL and ACS mRNA expression in RNA extracts of the stromal-vascular cell fraction (SVF) was also assessed. LPL mRNA was present in the SVF, but its expression did not change during starvation (Table 2). Furthermore, ACS was not expressed in the SVF fraction, suggesting that the cells expressing LPL mRNA in the SVF are mainly preadipocytes at a very early stage of differentiation (Amir et al. 1991).

LPL synthesis.  As depicted in Figure 2 and Table 3, starvation for 1 d did not affect rates of LPL synthesis relative to rates of total protein synthesis, but significantly decreased adipocyte total protein synthesis to 56% of control values. Therefore the decrease in total extractable LPL activity to 36% of control values after 1 d of starvation appeared to be mainly accounted for by the decrease in total protein synthesis. However, the decrease in HR-LPL activity (to 19% of control) was proportionately greater than the decrease in total extractable LPL activity and could not be explained at the level of LPL synthesis.

View larger version (89K): [in this window] [in a new window]   Fig 2. Lipoprotein lipase (LPL) synthesis in adipocytes of fed rats and rats starved for 1 or 3 d. Adipocytes were isolated from 4-mo-old (~420 g) rats that had been starved overnight (1 d), or controls that had had free access to food (fed) until killing at 1100 h. After a 30-min preincubation with 3.5 nmol/L insulin, cells were pulse-labeled with 35S-Express label (35S- met and cys) for 30 min and immediately homogenized. Equal trichloroacetic acid (TCA)-precipitable counts were used for immunoprecipitation of cellular LPL from fed and starved rat adipocytes, followed by SDS-PAGE. A fluorgraph representative of at least three independent replications of the experiment (Table 3) was made. The figure shows a fluorograph of one experiment representative of the four performed. Ctl, control [immunoprecipitation performed with nonimmune immunoglobulin G (IgG)].

Starvation for 3 d decreased relative rates of LPL synthesis to 44 ± 1% of control (n = 3, paired comparisons to control-fed levels, Table 3). Total adipocyte protein synthesis after 3 d of starvation also decreased to 44 ± 5% (n = 3) of fed controls. Thus the combined effects of decreased total protein synthesis and relative rates of LPL synthesis appeared to largely explain the decrease in total extractable and heparin-releasable LPL activity to 24 and 15% of control values, respectively (Table 1).

Effect of insulin on LPL synthesis in adipocytes of fed rats and rats starved for 1 d.  Because pulse-labeling was conducted in the presence of insulin, it was of importance to determine whether insulin had an acute stimulatory effect on LPL synthesis in adipocytes of either fed rats or those starved for 1 d. In these experiments, adipocytes from 6- to 8-wk-old rats were used because they are more responsive to insulin than adipocytes of 4-mo-old rats (Fried et al. 1990). As shown in Table 4 and Figure 3, consistent with results obtained in 4-mo-old rats (Fig. 3), relative rates of LPL synthesis were similar in adipocytes isolated from younger rats that were fed or starved for 1 d, regardless of whether incubations were condcuted in the absence or presence of insulin. Insulin produced a small but significant stimulation of relative rates of LPL synthesis (+38 ± 15%). In these experiments, insulin also stimulated overall protein synthesis (1.6 ± 0.05-fold and 1.6 ± 0.1-fold in adipocytes from starved and fed groups, respectively). The combination of specific and nonspecific effects of insulin resulted in an overall stimulation of LPL synthesis by 2.3 ± 0.3 (fed) and 2.3 ± 0.4-fold (starved). Thus, the inclusion of insulin during the pulse-labeling period in experiments comparing rates of LPL synthesis in fed rats and those starved for 1 d (Table 3) is unlikely to have influenced the result.

View larger version (71K): [in this window] [in a new window]   Fig 3. Effect of insulin on lipoprotein lipase (LPL) synthesis in adipocytes of rats that were fed or starved for 1 d.. Adipocytes were isolated from an 8-wk-old rat that had been fed or starved for 1 d and preincubated for 30 min in the presence or absence of 3.5 nmol/L insulin; 35S-Express label was then added and the incubation continued for 15 min. The figure shows a fluorograph of the immunoprecipiated LPL typical of the data summarized in Table 3. Ctrl, control immunoprecipitation.

LPL turnover.  The marked decrease in HR LPL activity after 1 d of starvation was not fully explained at the level of LPL synthesis. Therefore we investigated rates of adipocyte LPL degradation using a pulse-chase protocol. To enable comparison of the rates of LPL degradation relative to total protein degradation, LPL was immunoprecipitated from aliquots of fat cell homogenates containing equal counts of TCA-precipitable protein at each time point. Total protein degradation did not differ between adipocytes of fed and starved rats (data not shown). However, specific LPL degradation was faster in adipocytes of starved compared with fed rats (Fig. 4). The sum of cellular and secreted ³⁵S-LPL remaining after a 2-h chase period in adipocytes of fed rats was 85 ± 7% (n = 5) vs. 48 ± 8% (n = 4) in adipocytes of starved rats (P < 0.01, independent t test, Fig. 4). The estimated half-life of LPL was 2 h in adipocytes of starved and 5 h in those of fed rats. Even more strikingly, only 2 ± 1% (n = 4) compared with 12 ± 4 % (n = 5, P < 0.04 vs. starved) of the newly synthesized LPL was spontaneously released into the medium from adipocytes of rats starved for 1 d versus fed rats (Fig. 5). Thus, LPL degradation is more rapid in adipocytes of starved than fed rats, and LPL secretion is markedly decreased.

View larger version (16K): [in this window] [in a new window]   Fig 4. Degradation of newly synthesized biosynthetically labeled lipoprotein lipase (LPL) in adipocytes of rats that were fed or starved for 1 d. The figure shows a semilog plot of the disappearance of 35S-LPL (sum of immunoprecipitated cellular and secreted 35S-LPL) in pulse-chase experiments. After pulse-labeling for 15 or 30 min as described in the legend to Figure 2, aliquots of cells were immediately homogenized (pulse) or washed, then incubated in the presence of excess methionine and cysteine, but without insulin, for up to 135 min. Labeled LPL was immunoprecipitated from 0.5-mL aliquots of media samples or quantities of cell homogenates containing 106 counts per minute of 35S. Samples were processed by SDS-PAGE and fluorography and the intensity of the bands was quantified by densitometry. The values for cells and media were summed to calculate the rate of disappearance of the label. Data are means ± SEM of 2-4 independent values per time point. Lines show linear fit to the mean data.

View larger version (21K): [in this window] [in a new window]   Fig 5. Secretion of 35S-lipoprotein lipse (LPL) from adipocytes of rats that were fed or starved for 1 d. The percentage of the pulse-labeled LPL that was secreted into the medium at each time point of chase is plotted. To compare rates of LPL secretion during the chase period as a percentage of initial cellular values, densitometric valus for 35S-LPL in the cells and medium were normalized to the number of fat cells per milliliter in the original incubation. Data were calculated from the experiments described in the legend to Figure 4. Values are means ± SEM of the indicated numbers of independent experiments.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our data demonstrate that the mechanisms regulating rat adipose tissue LPL activity vary with the duration of starvation. After a long period of starvation, alterations in LPL mRNA expression contribute importantly to the marked decreases in total and heparin-releasable adipose tissue LPL activity. In contrast, after 1 d of starvation, adipocyte LPL mRNA levels and relative rates of LPL synthesis do not change, and the decline in heparin-releasable LPL activity is partially accounted for by a decrease in total protein synthesis. Additionally, in adipocytes of rats starved for 1 d vs. those of fed rats, more newly synthesized LPL is degraded, resulting in a dramatic decrease in the amount of newly synthesized LPL that is secreted. Thus with short-term starvation, LPL degradation is an important determinant of the amount of LPL available for secretion. With more severe starvation, pretranslational mechanisms become the predominant mechanism regulating the appearance of LPL activity in an HR pool.

Pretranslational regulation of LPL activity during prolonged starvation.  Our finding that LPL mRNA levels decrease with prolonged starvation is in good agreement with that of Ladu et al. (1991), who also used 4-mo-old male Wistar rats and found that starvation-induced changes in LPL mRNA were tightly correlated with the changes in LPL activity. It is apparent in the data from that study, as well as our own, that the correlation between LPL activity and mRNA levels is stronger after longer periods of starvation (3 or 6 d). Ladu et al. (1991) found a small (20%) significant decrease in adipose tissue LPL mRNA levels after 1 d of starvation, similar to the trend in this study (17%). It may be difficult to document a small decline in adipocyte LPL mRNA levels after overnight starvation because of the variability among rats in the timing of the last meal eaten and hence the actual length of the overnight (~1 d) starvation period.

There was a trend toward a decrease in adipocyte ACS mRNA with starvation. This effect was signficant in an independent experiment with 6-mo-old rats (Lee, J.-J., Smith, P. J. and Fried, S. K., unpublished observation). ACS gene expression is increased by insulin at the transcriptional level in 3T3-L1 adipocytes (Weiner et al. 1992) but is also increased by fatty acids (Amir et al. 1991). It is possible that during starvation, increases in fatty acids minimize the decline in ACS mRNA expression despite the fall in the plasma insulin. Further experiments will be required to assess whether the activity of ACS is an important determinant of rates of esterification of exogenous fatty acids in adipocytes during starvation.

In adipose tissue, LPL mRNA is expressed predominantly in adipocytes but also in the SVF, within preadipocytes. To facilitate comparisons of rates of adipocyte LPL biosynthesis with adipocyte mRNA levels, we assessed mRNA levels in RNA extracted from isolated adipocyte and SVF cell fractions as well as total adipose tissue. Unlike adipocyte LPL mRNA levels, SVF LPL mRNA levels were not influenced by starvation. However, the quantitative contribution of the SVF LPL mRNA to total tissue expression is probably minor. Thus measurement of total adipose tissue LPL mRNA levels closely reflects adipocyte levels and is adequate for most studies.

The hormonal/substrate mediators of starvation-induced downregulation of LPL mRNA expression remain to be elucidated. Insulin stabilizes LPL mRNA levels; thus the decrease in plasma insulin levels may be one important factor (Raynolds et al. 1990). Catecholamines, which are elevated in starvation, downregulate LPL mRNA expression (Raynolds et al. 1990), although reports are conflicting (Ong et al. 1992). Fatty acids may also play a role (Kirkland et al. 1994, Montalto and Bensadoun 1993).

Potential influences of starvation on total RNA levels and rates of protein synthesis must be considered in attempting to determine whether pretranslational mechanisms fully account for the observed changes in LPL activity. Kahn et al. (1989) estimated that RNA content per cell was decreased after starvation and that this mechanism explained the alterations in glucose transporter expression in fat cells during starvation and feeding. It is likely that alterations in total RNA content also contribute to the effects of starvation on overall protein synthesis (per cell) reported in this study. However, the nonquantitative nature of the RNA extraction procedure precludes drawing a firm conclusion on this issue. Additionally, it is possible that a decrease in the transport of amino acids into the fat cells from starved rats contributes to the decreased protein synthesis observed.

Effect of starvation on LPL synthesis.  Changes in the rate of LPL synthesis per fat cell during starvation can be due to nonspecific effects on total protein synthesis as well as to alterations in relative rates of LPL synthesis (as a percentage of total protein synthesis). In agreement with earlier studies (Lyons et al. 1980, Semb and Olivecrona 1989), we found that total protein synthesis is decreased by starvation and contributes to the decreases in LPL activity. In this study, the marked decrease (70 to 90%) in total extractable adipose tissue LPL activity (expressed per adipocyte) that occurred after a severe nutritional intervention (3 d of starvation) was due to the following combination of events: 1) a specific decrease in LPL mRNA levels (50 to 60% relative to total RNA), which accounted for the decreased rates of LPL biosynthesis (50% relative to total protein synthesis) and 2) a general decrease in adipocyte protein synthesis with starvation (50% on a per cell basis). However, after a less severe period of starvation (1 d), the relative abundance of LPL mRNA and relative rates of LPL synthesis were unchanged, but there was decrease in total protein synthesis per cell (40%). In contrast to these results, Oliver and Rogers (1993) found a 50% decrease in relative LPL synthesis as well as a 19% decrease in total protein synthesis in the adipose tissue of rats starved for 1 d compared with those that were fed (age not specified). The use of intact fragments of adipose tissue in that study (Oliver and Rogers 1993), compared with the isolated adipocytes in this study, may explain this discrepancy. Semb and Olivecrona (1989) found that adipocytes account for only 17% of total protein synthesis in guinea pig adipose tissue. If starvation has a greater effect on general protein synthesis in adipocytes vs. nonadipocytes, calculated rates of LPL synthesis relative to total tissue protein synthesis would be decreased, whereas rates of LPL synthesis relative to adipocyte protein synthesis would be unchanged. These difficulties in data analysis underline the importance of using isolated adipocytes for studies of the nutritional regulation of LPL metabolism.

The conclusion of this study, that relative rates of LPL synthesis are not affected by starvation for 1 d, contrasts with the conclusion of Doolittle et al. (1990) that starvation increases relative rates of LPL synthesis. However, it is difficult to compare the two studies. The earlier study compared LPL expression in adipose tissue of rats starved for 12 h with that of rats starved for 24 h and refed for 12 h (Doolittle et al. 1990). Their conclusion that starvation increased LPL mRNA and synthesis may be due to the use of intact adipose tissue fragments versus isolated fat cells, as discussed above, as well as the specific refeeding paradigm used (Doolittle et al. 1990). However, using a similar refeeding protocol, Oliver and Rogers (1993) did not find differences in relative rates of LPL synthesis in adipose tissue of fed and refed rats. Further studies should include assessment of the time course of the changes in adipocyte LPL expression during starvation and refeeding to reconcile these discrepancies.

We also considered the possibility that including insulin during the adipocyte pulse-labeling had influenced the results. We used young rats in the study to maximize our chance of detecting a significant insulin effect (Fried et al. 1990). We found that insulin produced a small (~30%) but consistent stimulation of LPL synthesis in freshly isolated adipocytes of both fed and starved rats. These data confirm and extend results in primary cultures of adipocytes (Ong et al. 1988), but contrast with results in 3T3-L1 adipocytes in which insulin effects on LPL activity were not associated with changes in LPL synthetic rate (Semenkovich et al. 1989).

Post-translational regulation of adipocyte LPL during starvation.  There is only one previous paper in the literature examining posttranslational LPL degradation as a function of nutritional state (Doolittle et al. 1990). They showed that, compared with refeeding, 12 h of starvation was associated with an increase in LPL degradation measured in adipose tissue fragments (Doolittle et al. 1990). Our studies show that the rate of LPL degradation is increased in starved compared with fed controls. Furthermore, by using isolated adipocytes rather than intact adipose tissue fragments, we could directly assess rates of LPL secretion. Measurement of LPL secretion is not possible with studies of adipose tissue fragments because the intact vasculature retains LPL released from the adipocyte. This work is the first to establish that alterations in the rate of spontaneous LPL secretion occur in concert with alterations in LPL degradation in rat adipocytes.

The half-life of LPL in this study of freshly isolated adipocytes (~2 h in starved rats, ~5 h in fed rats) is longer than that reported by others using isolated adipocytes that had been placed in primary culture overnight (~1 h) (Ong et al. 1992, Simsolo et al. 1992). The reason for this discrepancy is unknown, but it will be important to investigate the hormonal and metabolic factors regulating LPL turnover in fresh compared with cultured adipocytes.

It is also as yet unclear which hormones or substrates mediate the effect of starvation of LPL synthesis, degradation/secretion. In the fed state, LPL degradation may be slowed by the combination of insulin and glucocorticoid, similar to results from studies of human adipose tissue in vitro (Appel and Fried 1992). Catecholamines and insulin are also likely to play roles, but the literature is still inconsistent with regard to specific effects of these hormones on different steps in LPL synthesis and secretion (Enerback and Gimble 1993). It does not appear that starvation affects the translation of LPL mRNA, as reported for treatment with epinephrine (Ong et al. 1992, Ranganathan et al. 1997) because, in our studies, there was a parallel decline in LPL mRNA levels and rates of LPL biosynthesis.

Alterations in LPL specific activity have been reported to be at least a partial explanation for the changes in LPL activity that occur after starvation (Bergo et al. 1996, Doolittle et al. 1990). Bergo et al. (1996) recently reported that LPL activity decreased by 70-80% and LPL mass decreased by 20-40% in the adipose tissue of rats starved for 1 d compared with fed rats (Bergo et al. 1996). The decreased LPL specific activity in adipose tissue of starved rats was associated with an increase in monomeric (inactive) LPL. We did not directly measure LPL specific activity in this study; thus we cannot rule out its contribution to the decreased LPL activity. However, the alterations in LPL activity after both 1 and 3 d of starvation appear to be explained largely by the combination of specific alterations in LPL synthesis (associated with changes in LPL mRNA expression), alterations in LPL degradation (after 1 d of starvation) and a generalized decrease in adipocyte protein synthesis.

In summary, this work clarifies some discrepancies in the literature concerning whether starvation alters LPL mRNA levels and rates of LPL synthesis at the level of the adipocytes. After longer periods of starvation (3 d), the decrease in LPL activity results from a combination of a specific decrease in LPL mRNA levels and LPL synthesis and a decrease in overall protein synthesis. After relatively short periods of starvation (overnight), the decrease in extracellular, HR LPL activity appears to be accounted for by an increase in the rate of LPL degradation that leads to a decrease in the LPL activity secreted. Our results underline the utility of using freshly isolated adipocytes for studies of the regulation of LPL biosynthesis and secretion. Furthermore, these data provide a physiologically important example supporting the hypothesis that degradation is an important point in the regulation of LPL activity (Cisar et al. 1989, Semb and Olivecrona 1989).

	ACKNOWLEDGMENTS

We thank Colleen Russell for careful review of the manuscript and Robert Petersen for technical assistance.

	FOOTNOTES

Manuscript received 6 August 1997. Initial reviews completed 26 September 1997. Revision accepted 9 February 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Appel B., Fried S. K. Effects of insulin and dexamethasone on lipoprotein lipase in human adipose tissue. Am. J. Physiol. 1992; 262:E695-E699[Abstract/Free Full Text]
• Amir E.-Z., Ailhaud G., Grimaldi P. Regulation of adipose cell differentiation. II. Kinetics of induction of the aP2 gene by fatty acids and modulation by dexamethasone. J. Lipid Res. 1991; 32:1457-1463[Abstract]
• Bergo M., Olivecrona G., Olivecrona T. Forms of lipoprotein lipase in rat tissues: in adipose tissue the proportion of inactive lipase increases on fasting. Biochem. J. 1996; 313:893-898
• Braun J.E.A., Severson D. L. Regulation of the synthesis, processing and translocation of lipoprotein lipase. Biochem. J. 1992; 287:337-347
• Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987; 162:156-159[Medline]
• Cisar L. A., Hoogewerf A. J., Cupp M., Rapport C. A., Bensadoun A. Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparin sulfate proteoglycans is necessary for degradation. J. Biol. Chem. 1989; 264:1767-1774[Abstract/Free Full Text]
• DiGirolamo M., Mendlinger S., Fertig J. A simple method to determine fat cell size and number in four mammalian species. Am. J. Physiol. 1971; 221:850-858[Free Full Text]
• Doolittle M. H., Ben-Zeev O., Elovson J., Martin D., Kirchgessner T. G. The response of lipoprotein lipase to feeding and fasting. Evidence for posttranslational regulation. J. Biol. Chem. 1990; 265:4570-4577[Abstract/Free Full Text]
• Enerback S., Gimble J. M. Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level. Biochem. Biophys. Acta 1993; 1169:107-125[Medline]
• Fried S. K., Russell C. D., Grauso N. L., Brolin R. E. Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men. J. Clin. Invest. 1993; 92:2191-2198
• Fried S. K., Turkenkopf I. J., Goldberg I. J., Doolittle M. H., Kirchgessner T. G., Schotz M. C., Johnson P. R., Greenwood M.R.C. Mechanisms of increased lipoprotein lipase in fat cells of obese Zucker rats. Am. J .Physiol. 1992; 261:E653-E660
• Fried S. K., Velazquez N., Nobel J. Nutrition-induced variations in responsiveness to insulin effects on lipoprotein lipase activity in isolated rat fat cells. J. Nutr. 1990; 120:1087-1095
• Fried S. K., Zechner R. Effects of cachectin/tumor necrosis factor on human adipose tissue lipoprotein lipase activity, mRNA levels, and biosynthesis. J. Lipid Res. 1989; 30:1917-1923[Abstract]
• Hirsch J., Gallian E. Methods for the determination of adipose cell size in man and animals. J. Lipid Res. 1968; 9:110-119[Abstract]
• Iverius P., Brunzell J. D. Human adipose tissue lipoprotein lipase: changes with feeding and relation to postheparin plasma enzyme. Am. J. Physiol. 1985; 249:E107-E114[Abstract/Free Full Text]
• Kahn B. B., Cushman S. W., Flier J. S. Regulation of glucose transporter-specific mRNA levels in rat adipose cells with fasting and refeeding. J. Clin. Invest. 1989; 83:199-204
• Kirkland J. L., Hollenberg C. H., Kindeler S., Roncari D. Long-chain fatty acids decrease lipoprotein lipase activity of cultured rat adipocyte precursors. Metabolism 1994; 43:144-151[Medline]
• Ladu M. J., Kapsas H., Palmer W. K. Regulation of lipoprotein lipase in adipose and muscle tissues during fasting. Am. J. Physiol. 1991; 260:R953-R959[Abstract/Free Full Text]
• Lyons R. T., Nordeen S. K., Young D. A. Effects of fasting and insulin administration on polyribosome formation in rat epididymal fat cells. J. Biol. Chem. 1980; 255:6330-6334[Abstract/Free Full Text]
• Montalto M. B., Bensadoun A. Lipoprotein lipase synthesis and secretion: effects of concentration and type of fatty acids in adipocyte cell culture. J. Lipid Res. 1993; 34:397-407[Abstract]
• Oliver J. D., Rogers M. P. Stimulation of lipoprotein lipase synthesis by refeeding, insulin and dexamethasone. Biochem. J. 1993; 292:525-530
• Ong J. M., Saffari B., Simsolo R. B., Kern P. A. Epinephrine inhibits lipoprotein lipase gene expression in rat adipocytes through multiple steps in posttranscriptional processing. Mol. Endocrin. 1992; 6:61-69[Abstract/Free Full Text]
• Ong J. M., Kirchgessner T. G., Schotz M. C., Kern P. A. Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes. J. Biol. Chem. 1988; 253:12933-12938
• Ranganathan G., Vu D., Kern P. A. Translational regulation of liporotein lipase by epinephrine involves a trans-acting binding protein interacting with the 3-untranslated region. J. Biol. Chem. 1997; 272:2515-2519[Abstract/Free Full Text]
• Raynolds M. V., Awald P. D., Gordon D. F., Gutierrez-Hartmann A., Rule D. C., Wood W. M., Eckel R. H. Lipoprotein lipase gene expession in rat adipocytes is regulated by isoproterenol and insulin through different mechanisms. Mol. Endocrinol. 1990; 4:1416-1422[Abstract/Free Full Text]
• Semb H., Olivecrona T. Two different mechanisms are involved in the nutritional regulation of lipoprotein lipase in guinea pig adipose tissue. Biochem. J. 1989; 262:505-511[Medline]
• Semenkovich C. F., Wims M., Noe L., Etienne J., Chan L. Insulin regulation of lipoprotein lipase activity in 3T3-L1 adipocytes is mediated at posttranscriptional and posttranslational levels. J. Biol. Chem. 1989; 264:9030-9038[Abstract/Free Full Text]
• Shimomura I., Takahashi M., Tokunaga K., Keno Y., Nakamura T., Yamashita S., Takemura K., Yamamoto T., Funahashi T., Matsuzawa Y. Rapid enhancement of acyl-CoA synthetase, LPL and GLUT-4 mRNAs in adipose tissue of VMH rats. Am. J. Physiol. 1996; 270:E995-E1002
• Simsolo R. B., Ong J. M., Kern P. A. Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes. J. Lipid Res. 1992; 33:1777-1784[Abstract]
• Tukey, J.W. (1977) Exploratory Data Analysis. Addison-Wesley, Reading, MA.
• Wardzala L. J., Cushman S. W. Mechanism of insulin action on glucose transport in the isolated rat adipose cell. J. Biol. Chem. 1978; 253:8002-8005[Free Full Text]
• Weiner F. R., Smith P. J., Wertheimer S., Rubin C. S. Regulation of gene expression by insulin and tumor necrosis factor in 3T3-L1 cells. Modulation of the transcription of the genes encoding acyl-CoA synthetase and stearoyl-CoA desaturase. J. Biol. Chem. 1992; 266:23525-23528[Abstract/Free Full Text]

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