Antagonistic Effects of Vitamin D and Parathyroid Hormone on Lipoprotein Lipase in Cultured Adipocytes

Transcription

1 J Am Soc Nephrol 10: , 1999 Antagonistic Effects of Vitamin D and Parathyroid Hormone on Lipoprotein Lipase in Cultured Adipocytes UWE QUERFELD,* MICHAEL M. HOFFMANN, GÜNTER KLAUS, FRANK EIFINGER,* MARION ACKERSCHOTT,* DIETRICH MICHALK,* and PHILIP A. KERN *University Children s Hospital, Cologne, Germany; Department of Clinical Chemistry, Albert-Ludwigs- Universität, Freiburg, Germany; University Children s Hospital, Marburg, Germany; Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas. Abstract. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) (calcitriol) and parathyroid hormone (PTH) on synthesis and secretion of lipoprotein lipase (LPL) were studied in 3T3-L1 adipocytes. Expression of the vitamin D receptor was demonstrated by saturation kinetics with radiolabeled calcitriol. Incubation with calcitriol (10 8 M) for up to 4 d resulted in a time-dependent significant increase in heparinreleasable LPL activity (LPLa) accompanied by a significant increase in LPL mrna. In contrast, incubation with intact (1-84) PTH (10 6 to 10 9 M) produced a time- and dosedependent significant decrease in LPLa, but no change in LPL mrna. The effect of PTH (24-h incubation, 10 8 M) could be prevented by the calcium channel blocker verapamil. Coincubation with both calcitriol and PTH at equimolar concentration (10 8 M) resulted in an increase in LPLa and LPL mrna. These data indicate an antagonistic role for calcitriol and PTH in the regulation of LPL, possibly mediated by intracellular calcium, which may contribute to the alterations in lipoprotein metabolism occurring in uremia. Triglyceride-rich lipoproteins are removed from circulating plasma mainly after enzymatic hydrolysis by lipoprotein lipase (LPL), which is located at the capillary endothelium of adipose tissue, skeletal muscle, heart, and other tissues. Adipocytes are a major source of plasmatically active LPL, and in response to dietary need and nutritional supply, LPL synthesis in adipocytes is under control by insulin and other metabolic hormones (1 4). Previous studies of LPL regulation in adipocytes have provided important information for the understanding of the central role of this enzyme in lipoprotein metabolism and atherogenesis (5 7). In the wake of LPL-mediated triglyceride hydrolysis, surface material, i.e., phospholipids and apolipoproteins, is transferred into the plasma pool of HDL. If the activity of LPL is diminished, one would anticipate incomplete clearance of lipoproteins with accumulation of intermediate density lipoproteins enriched in triglycerides and with abnormal apolipoprotein composition, increased levels of total triglycerides, and diminished formation of HDL particles. All of these abnormalities are frequently found in patients with chronic renal failure Received December 8, Accepted April 1, This study was presented in part at the 28th Annual Meeting of the American Society of Nephrology, San Diego, CA, November 5 8, Correspondence to Dr. Uwe Querfeld, University Children s Hospital, Joseph- Stelzmann-Strasse 9, Cologne, Germany. Phone: ; Fax: ; Uwe.Querfeld@uni-koeln.de / Journal of the American Society of Nephrology Copyright 1999 by the American Society of Nephrology (CRF) and are believed to contribute to the highly increased risk for atherosclerotic complications in this population (8,9). Most studies agree that impaired catabolism of triglyceriderich lipoproteins is the main pathogenic factor in uremic dyslipidemia (10). Studies in patients with CRF have indeed demonstrated that LPL activity (LPLa) in adipose tissue (11) and post-heparin plasma (10,12) is decreased. The decrease in plasma post-heparin LPLa is correlated positively with triglyceride levels and inversely with HDL-cholesterol levels in dialysis patients (13,14). Furthermore, 5/6 nephrectomy in rats is followed by a progressive increase in plasma triglycerides, which is correlated with a steady decline in LPLa and LPL mrna levels in heart, muscle, and adipose tissue (15). CRF is also characterized by elevated plasma levels of parathyroid hormone (PTH) and diminished levels of the active vitamin D metabolite 1,25(OH) 2 D 3 (calcitriol). It has long been hypothesized that PTH could be the mediator of uremic dyslipidemia (16). Either independently or by regulating the synthesis of PTH (17), calcitriol could also affect lipoprotein metabolism in CRF. Normalization of PTH levels and a beneficial effect on plasma lipoproteins (lowering of triglycerides, increase in HDL-cholesterol) were indeed observed in uremic patients treated with vitamin D analogues (18 20). We therefore investigated the effects of calcitriol and PTH on LPL synthesis and secretion in the murine preadipocyte cell line 3T3-L1, a widely used model for the study of adipocyte LPL regulation (6). The results of our study indicate that 1,25(OH) 2 D 3 and PTH can independently affect lipoprotein metabolism by a direct regulation of adipocyte LPL production.

2 J Am Soc Nephrol 10: , 1999 Vitamin D, PTH, and LPL in Adipocytes 2159 Materials and Methods Cell Culture 3T3-L1 cells were obtained from American Type Culture Collection (Rockville, MD), and fibroblasts (preadipocytes) were grown to confluence in Dulbecco s modified Eagle s medium (obtained from Life Technologies BRL) supplemented with glucose, 10% fetal calf serum, and penicillin/streptomycin (all purchased from Sigma, Deisenhofen, Germany). Medium was changed every 2 to 3 d. Cells were differentiated into adipocytes by the addition of 20% fetal calf serum, 1 M dexamethasone, 0.5 mm methyl-isobutylxanthine, and 10 g/ml insulin (all from Sigma). After 48 h, medium was changed to Dulbecco s modified Eagle s medium plus insulin (50 ng/ml) for an additional 7 d, during which cells developed into mature adipocytes with typical histologic appearance. After this period cells were kept in insulin-free medium. For the experiments, calcitriol (Rocaltrol, obtained from Hoffman-LaRoche, Basel, Switzerland) or intact bovine PTH (1-84; obtained from Sigma) was added at the times and concentrations indicated. To evaluate the effect of calcium channel blockers, verapamil (verapamil hydrochloride obtained from Sigma) was added to the medium (at a concentration of 4 and 40 M, respectively) just before the addition of PTH. Media (including any additions) were changed every 2 to 3 d and always 24 h before LPL measurements. Vitamin D Receptor For the quantification of vitamin D receptor (VDR) expression, suspensions of untreated differentiated adipocytes (10 7 cells/ml) were homogenized in 0.4 M KTED buffer on ice. A purified fraction was prepared by centrifugation at 205,000 g for 30 min using a Ti-50 rotor (Beckman Instruments, Fullerton, CA). The supernatant was used for binding studies. Saturation analysis according to Scatchard (21) was carried out as described (22). Aliquots (100 l, protein concentration 0.5 to 1.2 mg/ml) were incubated for 16 h at 4 C with increasing concentrations (0.1 to 7.0 nm) of [ 3 H]-1,25(OH) 2 D 3 in the absence or presence of 100-fold molar excess of radioinert 1,25(OH) 2 D 3. Bound [ 3 H]- 1,25(OH) 2 D 3 was determined using the hydroxylapatite assay (23). Quantification of mrna Total RNA was extracted according to the method of Chomczynski and Sacchi (24). The final RNA preparation was dissolved in 300 l of RNase-free water and stored at 80 C. For the slot blot experiments, a Bio-Dot SF Microfiltration Apparatus (Bio-Rad Laboratories, Munich, Germany) was used. To measure the concentrations of the specific mrna for LPL, glycerinaldehyde-dehydrogenase (GAPDH) and the PTH-receptor (PTH-R) in parallel, 100 l of each RNA was transferred to a Hybond-N membrane (Amersham, Buckinghamshire, United Kingdom). After the transfer with the help of a vacuum device, the RNA was fixed on the membrane by baking for 2 h at 80 C. For hybridization, the membrane was incubated in a solution consisting of 6 SSC, 0.5% sodium dodecyl sulfate, 5 Denhardt s solution, and 100 g/ml denatured salmon sperm DNA. The following cdna probes were used: human LPL (complete coding region; generous gift from Dr. M. Schotz, Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, CA), human GAPDH (complete coding region), and rat PTH-R (complete coding region; generous gift from Dr. G. Gross, National Research Center for Biotechnology, Braunschweig, Germany). The cdna probes were radiolabeled by using a random priming kit (Boehringer Mannheim) and added after 1 h of preincubation. The hybridization took 16 h at 65 C. The blots were washed with 2 SSC until the background disappeared. x-ray films were exposed for 12 to 36 h at 80 C. The autoradiography of the blots was analyzed by using the ScanPack hard- and software (Biometra, Göttingen, Germany). Each blot was analyzed three times. Quantification of LPLa LPLa was measured in medium before and 45 min after the addition of heparin (10 U/ml) using a stable 3 H-triolein-lecithin emulsion prepared in glycerol (25). After incubation of media samples with this substrate for 30 min at 37 C, the reaction was stopped by adding Belfrage-Vaughn solution (26). Labeled free fatty acids (FFA) liberated by the reaction were measured by scintillation counting, and activity was expressed as the amount of FFA generated per hour (nmol FFA/h per mg protein). All assays were performed in quadruplicate. Cells were denatured with 0.1 M NaOH for quantification of protein concentration by the BCA protein assay (Pierce, Rockford, IL). Statistical Analyses For comparison of LPLa and mrna in treated cells versus controls, each experiment consisted of separate measurements of at least three (and up to eight) different cell culture dishes for every condition examined. Data were expressed as mean SD. For statistical comparison, ANOVA followed by the Bonferroni t test was used to test the significance of changes in treated cells versus untreated controls. Results Vitamin D Receptor Mature adipocytes (day 10 after differentiation) expressed the VDR (5219 receptors/cell; K d ) (Figure 1). This concentration was in the order of magnitude described previously for cartilage and bone cells (22). Effect of Calcitriol on LPL Production by 3T3-L1 Adipocytes in Culture A continuous increase in LPLa could be demonstrated in the media of cells cultured for up to 34 d (Figure 2). Heparinreleasable LPLa in the medium was about fourfold compared Figure 1. Specific binding of [ 3 H]-1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) to adipocytes. (Left) Addition of free [ 3 H]- 1,25(OH) 2 D 3 to cultured mature 3T3-L1 adipocytes results in rapid increase of the bound fraction (high-affinity binding). (Right) Scatchard plot of the data gives an estimation of the receptors per cell.

3 2160 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Table 1. Effect of calcitriol and PTH on LPL mrna levels in adipocytes a Agent Treated Control Calcitriol b PTH d e e e 100 Calcitriol PTH f c 100 Figure 2. Heparin-releasable lipoprotein lipase (LPL) in long-term culture. After differentiation into mature adipocytes, cells were cultured for up to 3 wk (see Materials and Methods). Heparin-releasable LPL activity (bars indicate means SEM of three experiments) was measured every 3 d in the medium (24 h after exchange of medium) and increased continuously, indicating constitutive secretion of LPL by viable cells. Data were pooled from three experiments. a PTH, parathyroid hormone; LPL, lipoprotein lipase. b Mean ratio of LPL/GAPDH mrna levels (percentage of untreated control cells) after 3doftreatment with calcitriol (10 8 M) (three experiments). c P d Mean ratio of LPL/GAPDH mrna levels (percentage of untreated control cells) after 4 d of treatment with PTH at concentrations of 10 8 M (six experiments) and 10 7 to 10 9 M (two experiments), respectively. e Not significant. f Mean ratio of LPL/GAPDH mrna levels (percentage of untreated control cells) after 48 h of incubation with calcitriol (10 8 M) and PTH (10 8 M) (two experiments). to controls without heparin, and could be completely inhibited by the addition of 1 M NaCl to the assay, indicating the absence of other lipases (data not shown). On day 24 in culture (16 d after differentiation), cells were treated with calcitriol (10 8 M) for 1 to 4 d, and heparinreleasable LPLa was measured in the medium. Compared to untreated controls, LPLa was increased by 36 31% (NS) after 24 h of incubation, and significantly (P 0.05) increased by 72 17, 71 19, and 48 11% after 48, 72, and 96 h, respectively, of incubation (Figure 3). Calcitriol (10 8 M;3dofincubation) significantly increased LPL gene expression, as indicated by an increase in the LPL/ GAPDH mrna ratio by a mean of 42% compared to controls (P 0.05) (Table 1). PTH Receptor PTH-R-mRNA could be readily detected in mature adipocytes, and the synthesis of the PTH-R could be stimulated by the addition of PTH (10 8 M) to the medium. The mrna ratio (PTH-R/GAPDH) increased by a mean of and % (P 0.05), respectively, after 24 and 96 h of incubation (two experiments). Effect of PTH on LPL Production On day 16 after differentiation, PTH (10 6 to 10 9 M) was added to the medium of mature adipocytes for 24 h. Mean LPLa decreased in a dose-dependent manner, and compared to untreated controls, this effect was significant (P 0.05) at concentrations from 10 8 to 10 6 M (Figure 4). Figure 3. Time-dependent effect of calcitriol on LPL activity in 3T3-L1 cells. Heparin-releasable LPL activity was measured in the medium after incubation of cells with calcitriol (10 8 M) for 24 to 96 h. Data (bars indicate means SD) are expressed as percentage of untreated control cells. Data were pooled from four experiments. *P Figure 4. Dose-dependent effect of parathyroid hormone (PTH) on LPL activity in 3T3-L1 cells. Heparin-releasable LPL activity was measured in the medium after incubation of cells with PTH (10 9 to 10 6 M) for 48 h. Data (bars indicate means SD) are expressed as percentage of untreated control cells. Data were pooled from three experiments. *P 0.05.

4 J Am Soc Nephrol 10: , 1999 Vitamin D, PTH, and LPL in Adipocytes 2161 We then studied the effects of PTH over 4 d in culture. When PTH (10 8 M) was added to the medium, a significant decrease of LPLa could be observed after 24 h ( 41 10%); LPLa could be further suppressed by incubation for 48 h ( 53 34%), 72 h ( 80 36%), and 96 h ( 83 24%), respectively (Figure 5). To determine whether this effect was due to suppression of LPL synthesis in adipocytes, LPL mrna was quantified by slot blot analysis (Figure 6A). After 48 h of treatment with PTH (10 6 to 10 9 M), no significant change in the mrna ratio LPL/GAPDH was detected (Table 1). To study whether the suppressive effect of PTH on LPL production could be inhibited by calcium channel blockers, cells were coincubated with verapamil and PTH (10 8 M) for 24 h. LPLa was reduced to 75 1% of controls in PTH-treated cells (P 0.05). However, LPLa was not significantly different from untreated controls when cells were coincubated with PTH (10 8 M) and verapamil at a concentration of 4 and 40 M (Table 2). Combined Effects To study the combined effects of calcitriol and PTH on adipocyte LPL, cells were incubated simultaneously with both hormones at a concentration of 10 8 M for 1 to 3 d. This resulted in an increase of LPLa by 5 32% after 24 h (NS), % after 48 h (P 0.05), and % after 72 h (P 0.05), respectively, compared to untreated control cells. This was accompanied by an increase in LPL mrna levels (LPL/GAPDH mrna ratio) by approximately 22% (Table 1, Figure 6B). Figure 5. Time-dependent effect of PTH on LPL activity in 3T3-L1 cells. Heparin-releasable LPL activity was measured in the medium after incubation of cells with PTH (10 8 M) for 24 to 96 h. Data (bars indicate means SD) are expressed as percentage of untreated control cells. Data were pooled from five experiments. *P Discussion The present study shows that mature 3T3-L1 adipocytes express receptors for calcitriol and PTH and that these hormones affect LPL synthesis and secretion in an antagonistic manner. Calcitriol 1,25(OH) 2 D 3 the major biologically active vitamin D metabolite, is recognized as the principal mediator of vitamin D effects. Its receptor (VDR) has been described in several tissues, including kidney, bone, parathyroid glands, epidermis, cardiac muscle, circulating blood cells, etc. (27). In addition, we have demonstrated previously the presence of VDR in bone, cartilage (22), and 3T3-L1 cells (28), and the induction of differentiation and VDR expression by calcitriol in 3T3-L1 cells (28). Calcitriol is a multifunctional hormone with important regulatory functions in calcium homeostasis, cell differentiation, and the immune response. A large variety of genes are regulated by 1,25(OH) 2 D 3, and both transcriptional and posttranscriptional effects have been described (27). The present study shows that calcitriol produces a time-dependent stimulation of LPL secretion in cultured adipocytes, accompanied by an increase in LPL mrna. Extending our previous observation that calcitriol promotes adipocyte differentiation (28), the present data indicate a stimulatory role in adipocyte LPL regulation at the level of either gene transcription or mrna stabilization. Beneficial effects of calcitriol treatment on lipoproteins have been demonstrated in hemodialysis patients (18 20). However, there is only limited published evidence for a direct link between vitamin D and lipoprotein metabolism in healthy individuals in vivo. For example, a positive significant correlation between circulating 25-OH (reflecting vitamin D supply/intake) and apolipoprotein A-I and HDL-cholesterol levels, respectively, has been demonstrated in healthy adults (29). The present study confirms that adipose tissue is a target organ for PTH. In previous studies, PTH stimulated (intracellular) hormone-sensitive lipase in adipocytes, activated cgmp generation in human fat cell ghosts, and produced a dosedependent rise in cytosolic calcium levels in rat adipocytes (30). We demonstrate that PTH receptors are present in mature adipocytes and that their concentration is upregulated by treatment with PTH in vitro. PTH had a powerful dose- and time-dependent suppressive effect on LPL production by cultured adipocytes, but no effect on LPL transcription. These data suggest that PTH inhibits LPL by posttranslational modification, which might be explained by the known stimulatory effect of PTH on intracellular cgmp and camp. In previous studies by us, stimulation of camp by epinephrine decreased LPL in adipocytes via multiple posttranscriptional steps, including a decrease in LPL translation (3). Posttranslational modification of LPL by hormones and cytokines has also been described in vivo (5,31) and in several in vitro studies (1,32 34). Because low levels of calcitriol and an increase in PTH are part of the uremic syndrome, the results of the present study may contribute to the explanation of the pathophysiology of uremic dyslipidemia. The question Why is LPL activity reduced in renal failure? has puzzled many investigators. The presence of circulating inhibitor(s) in plasma seems an intuitive explanation in view of the accumulation of waste products in uremia, and an inhibitory effect of uremic serum on LPL activity has been described by several workers (35 38). A recent report has identified an apolipoprotein A-I-containing particle with pre- electrophoretic mobility as an inhibitor of LPL in normal and uremic plasma (39). In contrast, PTH does not seem to be a plasmatic inhibitor of LPL; PTH (if added in vitro to the LPL activity assay) does not interfere with the

5 2162 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , 1999 Figure 6. LPL synthesis in 3T3L1 adipocytes treated with calcitriol and PTH, assessed by slot blot autoradiography of mrna expression of LPL compared to GAPDH. (A) LPL and GAPDH mrna concentrations in cultured adipocytes (lanes 1 to 3) treated with calcitriol (10 8 M) or PTH(10 8 M) for 72 and 96 h, respectively. (B) LPL and GAPDH mrna concentrations in cultured adipocytes (lanes 1 to 3) treated with both calcitriol (10 8 M) and PTH (10 8 M) simultaneously for 48 h. Table 2. Effects of PTH and verapamil on LPLa in adipocytes a Agent Treated Control PTH b 75 1 c 100 PTH verapamil (4 M) d e 100 PTH verapamil (40 M) d e 100 a LPLa, lipoprotein lipase activity. b Heparin-releasable LPLa (percentage of controls) after 24 h of incubation with PTH (10 8 ). c P 0.05 (combined data from three experiments). d Heparin-releasable LPLa (percentage of controls) after 24 h of coincubation with PTH (10 8 ) and verapamil (4 M, 40 M). e Not significant. lipolytic activity of either human (plasmatic) LPL or purified bovine milk LPL (40). There is abundant experimental evidence indicating that chronic PTH excess is a main factor in the pathogenesis of uremic dyslipidemia (16). In animal experiments with established models of CRF, parathyroidectomy results in amelioration of hyperlipidemia in rats (41) and even in normalization of triglyceride levels, post-heparin lipolytic activity, and intravenous fat tolerance tests in dogs (42). Moreover, hypertriglyceridemia and hyperparathyroidism are associated not only in uremic patients, but also in patients with primary and secondary hyperparathyroidism (43). Previous work has also provided evidence that intact PTH produces a dose-dependent increase in intracellular calcium in adipocytes and that this can be prevented in vitro by the addition of the calcium channel blockers verapamil and nifedipine (30). Experiments in partially nephrectomized rats demonstrated that the increase in intracellular calcium found in uremic animals could be prevented by either parathyroidectomy or treatment with verapamil (44) and that treatment with verapamil prevented the CRF-induced abnormalities in lipid metabolism (45). Taken together, these studies indicate that uremic dyslipidemia is mediated by an excess of PTH and that on a cellular level, PTH acts by producing a sustained increase in intracellular calcium. The present study supports this concept since the effect of PTH on adipocyte LPLa could be prevented by the addition of verapamil. Interestingly, PTH has a similar suppressive effect on hepatic lipase. In rats with CRF, hepatic lipase mrna expression was significantly reduced, and parathyroidectomy or treatment with verapamil corrected impaired hepatic lipase expression (46). A similar downregulation of LPL gene expression has been demonstrated in experimental CRF in rats (15,47,48). Although adipocyte LPLa, plasmatic LPLa, and enzyme mass (11,49,50) are low in patients with CRF, to our knowledge LPL mrna expression in adipocytes has not been studied in human uremic dyslipidemia. We therefore do not know at present whether the suppressive effect of CRF in vivo occurs at the levels of transcription or by posttranscriptional modification of LPL, as suggested by our in vitro data. The results of the present study suggest that calcitriol and PTH may be physiologic regulators of adipocyte LPL synthesis and that the decrease of LPL activity in plasma and of releasable activity from adipose tissue in uremia (15) could at least in part be mediated by changes in circulating levels of these hormones, i.e., a decrease in calcitriol and excess of PTH. Although the concentrations of calcitriol and PTH used in these in vitro experiments differ from physiologic concentrations (approximately M), these hormones clearly have an antagonistic effect on adipocyte LPL. Interestingly, coincubation of adipocytes with both hormones at equal concentrations resulted in an increase in LPL activity and LPL transcription, indicating that under these in vitro conditions, calcitriol was able to override the PTH effect. One might speculate that in

6 J Am Soc Nephrol 10: , 1999 Vitamin D, PTH, and LPL in Adipocytes 2163 vivo the lack of even absence of calcitriol and the presence of high PTH concentrations may be required to significantly suppress adipocyte LPL production this constellation of hormonal changes is typical for CRF. In view of the profound metabolic disturbances of the uremic state, multiple other factors besides calcitriol and PTH are probably involved in the pathogenesis of uremic dyslipidemia (10). Thus, altered substrate characteristics of abnormally composed triglyceride-rich lipoproteins (51) and a downregulation of receptors for very low density lipoproteins (48,52) may contribute significantly to an accumulation of triglyceride-rich particles in plasma. Insulin is an essential regulator of adipocyte LPL synthesis, and the known insulin resistance of the uremic state could contribute to a diminished lipolytic activity. Insulin supplementation in uremic rats has indeed been shown to correct adipose tissue LPL (53). PTH could also indirectly affect LPL by its effect on insulin, e.g., by impairing pancreatic insulin release (54). In summary, the present study shows that calcitriol and PTH have antagonistic effects on adipocyte LPL in vitro. These data suggest a role for these multifunctional hormones in the regulation of lipoprotein metabolism and may help explain the changes in lipoproteins occurring in CRF. Acknowledgments The authors thank Mrs. A. Gradehand for expert technical assistance. References 1. Simsolo RB, Ong JM, Kern PA: Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes. J Lipid Res 33: , Ong JM, Simsolo RB, Saffari B, Kern PA: The regulation of lipoprotein lipase gene expression by dexamethasone in isolated rat adipocytes. Endocrinology 130: , Yukht A, Davis RC, Ong JM, Ranganathan G, Kern PA: Regulation of lipoprotein lipase translation by epinephrine in 3T3 L1 cells: Importance of the 3 untranslated region. J Clin Invest 96: , Eckel RH: Lipoprotein lipase: A multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 320: , Fried SK, Russell CD, Grauso NL, Brolin RE: Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men. J Clin Invest 92: , Eckel RH, Fujimoto WY, Brunzell JD: Development of lipoprotein lipase in cultured 3T3 L1 cells. Biochem Biophys Res Commun 78: , Olivecrona T, Liu G, Hultin M, Bengtsson-Olivecrona G: Regulation of lipoprotein lipase. Biochem Soc Trans 21: , Attman PO, Alaupovic P, Tavella M, Knight-Gibson C: Abnormal lipid and apolipoprotein composition of major lipoprotein density classes in patients with chronic renal failure. Nephrol Dial Transplant 11: 63 69, Ritz E, Querfeld U: Atherogenesis: Is it accelerated in uremia? Semin Dial 2: , Kaysen GA: Hyperlipidemia of chronic renal failure. Blood Purif 12: 60 67, Goldberg A, Sherrard DJ, Brunzell JD: Adipose tissue lipoprotein lipase in chronic hemodialysis: Role in plasma triglyceride metabolism. J Clin Endocrinol Metab 47: , Gupta KL, Majumdar S, Sakhuja V: Post-heparin lipolytic activity in acute and chronic renal failure. Ren Fail 16: , Chan MK, Persaud J, Varghese Z, Moorhead JF: Pathogenic roles of post-heparin lipases in lipid abnormalities in hemodialysis patients. Kidney Int 25: , Shoji T, Nishizawa Y, Nishitani H, Yamakawa M, Morii H: Impaired metabolism of high density lipoprotein in uremic patients. Kidney Int 41: , Vaziri ND, Liang K: Down-regulation of tissue lipoprotein lipase expression in experimental chronic renal failure. Kidney Int 50: , Massry SG, Akmal M: Lipid abnormalities, renal failure, and parathyroid hormone. Am J Med 87: 42N 44N, Silver J, Russell J, Sherwood LM: Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 82: , Lin SH, Lin YF, Lu KC, Diang LK, Chyr SH, Liao WK, Shieh SD: Effects of intravenous calcitriol on lipid profiles and glucose tolerance in uraemic patients with secondary hyperparathyroidism. Clin Sci 87: , Lind L, Lithell H, Wengle B, Wrege U, Ljunghall S: A pilot study of metabolic effects of intravenously given alpha-calcidol in patients with chronic renal failure. Scand J Urol Nephrol 22: , Yeksan M, Turk S, Polat M, Cigli A, Erdogan Y: Effects of 1,25 (OH) 2 D 3 treatment on lipid levels in uremic hemodialysis patients. Int J Artif Organs 15: , Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51: , Klaus G, von Eichel B, May T, Hügel U, Mayer H, Ritz E, Mehls O: Synergistic effects of parathyroid hormone and 1,25(OH) 2 D 3 on proliferation and vitamin D receptor expression of rat growth cartilage cells. Endocrinology 135: , Wecksler WR, Norman AW: A hydroxylapatite batch assay for the quantitation of 1,25-dihydroxyvitamin D3 receptor complexes. Anal Biochem 92: , Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem 162: , Nilsson-Ehle P, Schotz MC: A stable, radioactive substrate emulsion for assay of lipoprotein lipase. J Lipid Res 17: , Belfrage P, Vaughan M: Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J Lipid Res 10: , Reichel H, Koeffler HP, Norman AW: The role of the vitamin D endocrine system in health and disease. N Engl J Med 320: , Vu D, Ong JM, Clemens TL, Kern PA: 1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3 L1 cells in association with adipocyte differentiation. Endocrinology 137: , Auwerx J, Bouillon R, Kesteloot H: Relation between 25-hydroxyvitamin D3, apolipoprotein A-I, and high density lipoprotein cholesterol. Arterioscler Thromb 12: , 1992

7 2164 Journal of the American Society of Nephrology J Am Soc Nephrol 10: , Ni Z, Smogorzewski M, Massry SG: Effects of parathyroid hormone on cytosolic calcium of rat adipocytes. Endocrinology 135: , Doolittle MH, Ben Zeev O, Elovson J, Martin D, Kirchgessner TG: The response of lipoprotein lipase to feeding and fasting: Evidence for posttranslational regulation. J Biol Chem 265: , Ong JM, Saffari B, Simsolo RB, Kern PA: Epinephrine inhibits lipoprotein lipase gene expression in rat adipocytes through multiple steps in posttranscriptional processing. Mol Endocrinol 6: 61 69, Kamei Y, Kawada T, Fujita A, Etinne J, Noe L, Sugimoto E: Lipoprotein lipase enzyme expression in 3T3 L1 adipocytes is posttranscriptionally down-regulated by retinoic acid. Biochem Int 26: , Saffari B, Ong JM, Kern PA: Regulation of adipose tissue lipoprotein lipase gene expression by thyroid hormone in rats. J Lipid Res 33: , Murase T, Cattran DC, Rubenstein B, Steiner G: Inhibition of lipoprotein lipase by uremic plasma, a possible cause of hypertriglyceridemia. Metabolism 24: , McCaleb ML, Mevorach R, Freeman RB, Izzo MS, Lockwood DH: Induction of insulin resistance in normal adipose tissue by uremic human serum. Kidney Int 25: , Yukawa S, Tone Y, Sonobe M, Maeda T, Mimura K, Mune M, Fujiwara S, Tsujimoto K, Maeshima E, Saika Y: Study on the inhibitory effect of uremic plasma on lipoprotein lipase. Nippon Jinzo Gakkai Shi 34: , Bagdade JD, Yee E, Wilson DE, Shafrir : Hyperlipidemia in renal failure: Studies of plasma lipoproteins, hepatic triglyceride production, and tissue lipoprotein lipase in a chronically uremic rat model. J Lab Clin Med 91: , Cheung AK, Parker CJ, Ren K, Iverius P-H: Increased lipase inhibition in uremia: Identification of pre- -HDL as a major inhibitor in normal and uremic plasma. Kidney Int 49: , Arnadottir M, Nilsson Ehle P: Parathyroid hormone is not an inhibitor of lipoprotein lipase activity. Nephrol Dial Transplant 9: , Heuck CC, Liersch M, Ritz E, Stegmeier K, Wirth A, Mehls O: Hyperlipoproteinemia in experimental chronic renal insufficiency in the rat. Kidney Int 14: , Akmal M, Kasim SE, Soliman AR, Massry SG: Excess parathyroid hormone adversely affects lipid metabolism in chronic renal failure. Kidney Int 37: , Lacour B, Roullet J-B, Liagre A-M, Jorgetti V, Beyne P, Dubost C, Drüeke T: Serum lipoprotein disturbances in primary and secondary hyperparathyroidism in effects of parathyroidectomy. Am J Kidney Dis 8: , Ni Z, Smogorzewski M, Massry SG: Elevated cytosolic calcium of adipocytes in chronic renal failure. Kidney Int 47: , Akmal M, Perkins S, Kasim SE, Oh H-Y, Smogorzewski M, Massry SG: Verapamil prevents chronic renal failure-induced abnormalities in lipid metabolism. Am J Kidney Dis 22: , Klin M, Smogorzewski M, Ni Z, Zhang G, Massry SG: Abnormalities in hepatic lipase in chronic renal failure: Role of excess parathyroid hormone. J Clin Invest 97: , Vaziri ND, Wang XQ, Liang K: Secondary hyperparathyroidism downregulates lipoprotein lipase expression in chronic renal failure. Am J Physiol 273: F925 F930, Vaziri ND, Liang K: Down-regulation of VLDL receptor expression in chronic experimental renal failure. Kidney Int 51: , Joven J, Vilella E, Ahmad S, Cheung MC, Brunzell JD: Lipoprotein heterogeneity in end-stage renal disease. Kidney Int 43: , Attman PO, Samuelson O, Alaupovic P: Lipoprotein metabolism in chronic renal failure. Am J Kidney Dis 21: , Arnadottir M, Thysell H, Dallongeville J, Fruchart J-C, Nilsson- Ehle P: Evidence that reduced lipoprotein lipase activity is not a primary pathogenetic factor for hypertriglyceridemia in renal failure. Kidney Int 48: , Liang K, Oveisi F, Vaziri ND: Role of secondary hyperparathyroidism in the genesis of hypertriglyceridemia and VLDL receptor deficiency in chronic renal failure. Kidney Int 53: , Roullet JB, Lacour B, Yvert JP, Drueke T: Correction by insulin of disturbed TG-rich LP metabolism in rats with chronic renal failure. Am J Physiol 250: E373 E376, Fadda GZ, Akmal M, Premdas FH, Lipson LG, Massry SG: Insulin release from pancreas islets: Effects of CRF and excess PTH. Kidney Int 33: , 1987