Effect of gemfibrozil on adipose tissue and muscle lipoprotein lipase

Effect of Gemfibrozil

on Adipose

Rosa 6. Simsolo,

Tissue

John

and Muscle

Lipoprotein

Lipase

M. Ong, and Philip A. Kern

To better

understand the mechanism of action of gemfibrozil on plasma triglycerides, lipoprotein lipase (LPL) concentration was measured in adipose tissue and muscle of 16 hypertriglyceridemic patients before and after treatment with gemfibrozil for 6 weeks. The patients were divided into three groups based on clinical criteria as follows: group 1, hypertriglyceridemia without secondary factors; group 2, hypertriglyceridemia with diabetes; and group 3, hypertriglyceridemia with renal insufficiency. LPL activity, immunoreactive mass, synthetic rate, and mRNA levels were measured in the adipose tissue samples, and LPL activity and mass in the muscle samples. Serum triglyceride levels were decreased by 46% by gemfibroril, and patients demonstrated no change in diet, weight, or glycohemoglobin during the 6 weeks of treatment. Despite the decrease of blood triglyceride levels, there was no significant change in any measure of LPL either in adipose tissue or muscle. Although several patients demonstrated increases in muscle LPL activity, these changes were inconsistent and not statistically significant. Because there was no significant change in LPL, we conclude that gemfibrozil in these patients decreased circulating triglyceride levels predominantly by decreasing hepatic very-low-density lipoprotein (VLDL) secretion. Copyright 0 1993 by W.B. Saunders Company

L

IPOPROTEIN LIPASE (LPL) hydrolyzes the core of triglyceride-rich lipoproteins (very-low-density lipoproteins [VLDL] and chylomicrons) into free fatty acids (FFA) and monoacylglycerol.’ The two primary tissues that express LPL are adipose tissue and muscle, and FFA generated by LPL action are used for storage in the adipocyte and for energy in muscle. Abnormalities in LPL are present in common metabolic disorders such as diabetes”.” and chronic renal disease,” and the resulting hypertriglyceridemia and low high-density lipoprotein (HDL) levels contribute to the accelerated atherosclerosis seen in these conditions. Clofibrate and gemfibrozil are established drugs for decreasing serum triglyceride levels, and a number of studies have examined their mechanisms of action. In several studies, clofibrate increased LPL activity in both postheparin plasmaje7 and adipose tissue,” and this increase in LPL was likely an important mechanism for the decrease in circulating triglyceride levels after clofibrate treatment. In studies of gemfibrozil, there was a decrease in hepatic VLDL output.h,y apparently due to a decrease in triglyceride synthesis, which may in part be due to decreased adipose tissue lipolysis and FFA release.“’ However, gemfibrozil also increased the rate of clearance of triglyceriderich lipoproteins.h.” In one study where the effects of clofibrate and gemfibrozil were compared side by side, the fractional catabolic rate of VLDL was increased by clofibrate and gemfibrozil by 35% and 92%. respectively.h LPL-mediated triglyceride hydrolysis is the rate-limiting

step in the clearance of triglyceride-rich lipoproteins. and several studies have demonstrated increases in postheparin plasma LPL activity in patients treated with gemfibrozil.h.l?-I5 No studies have directly examined the elfects of gemfibroLPL in adipose tissue and muscle. which are the tissues from which most LPL is synthesized. In addition. previous studies measured only LPL activity and did not measure the LPL protein or mRNA. Thus the goal of this project was to determine whether gemfibrozil treatment of patients affected adipose tissue and muscle LPL. and if so, to determine the mechanism of this effect.

zil on

SUBJECTS AND METHODS Patients This was an open-label action of gemfibrozil.

study to examine

65 and had hypertriglyceridemia. triglyceride

level greater

of

with a fasting ( > 12 hours) blood

than 300 mg% (3.4 mmol/L).

were receiving no medications

Patients

known to elevate blood triglyceride

levels, such as thiazide diuretics. @blockers, isotretinoin,

the mechanism

All patients were between the ages of 31 and

and were not extremely

steroid hormones, and

obese (< 15OF

ideal body

weight). Patients were recruited who had hypertriglyceridemia

due

to (1) genetic factors (no secondary causes of hypertriglyceridemia). (2) diabetes, and (3) chronic renal disease. Characteristics Patients criteria

of the study patients

in group

I (genetic

are shown

hyperlipidemia)

in Table

conformed

1.

to the

above and had no evidence of diabetes or renal disease as

manifested by the initial fasting laboratory

tests. Patients in group 2

(hyperlipidemia

to the above criteria. and

and diabetes) conformed

all had type II diabetes.

They were in a clinically stable state of

glycemic control with either diet. oral agents, or insulin, and had a

From the Department of Medicine, Division of Endocrinology, Cedars-Sinai Medical Center, Los Angeles, CA. Submitted November 7, 1992; accepted Januaty 27. 1993. Supported by a grantfrom Parke-Davis/ Warner Lambert. A Career Development Award from the Juvenile Diabetes Foundation (J.M. 0. J, and National institutes of Health Grant No. DK 39176 (P.A. K. ). Conducted during the tenure of an Established Investigatorship from the American Heart Association (P.A.K.). Address reprint requests to Philip A. Kern, MD, Division of Endocrinology, Becker 131. Cedars-Sinai Medical Center, 8700 Beverlv Blvd, Los Angeles, CA 90048. Copyright 0 1993 by W.B. Saunders Company 0026-049519314211-0018$03.00/0 1486

level of less than t1.5. Patients in group 2 did not have clinically significant renal disease (calculated creatinine clearance, > 50 mL/min). Patients in group 3 (hyperlipidemia with renal disease) conformed to the above criteria, but had chronic renal insufficiency (calculated creatinine clearance. < 20 mL/min). Acceptable patients were clinically stable and were not suffering from acute changes in renal function. No patients were renal transplant recipients. but two were on long-term hemodialysis and one was on long-term ambulatory peritoneal dialysis. In the patients on hemodialysis, both sets of biopsies were performed the day after hemodialysis. Patients who agreed to participate and who fulfilled the criteria outlined above were taken off any lipid-lowering drugs for at least I glycosylated hemoglobin

Metabolism, Vol42, No 11 (November), 1993: pp 1466-1491

1487

EFFECT OF GEMFIBROZIL ON LPL

Table 1. Characteristics of Patients Glycosylated Body Mass

Triglycerides

Cholesterol

Index

(mg/dL)

(mg/dL)

C

G

Group 1: hypertriglyceridemia Mean SEM

C (n =

G

C

Hemoglobin

GlUCOSe (mg/dU G

1%)

C

G

1,735

659

401

293

89

93

1.3

658

293

79

19

10

9

27.7

27.9

649

375

287

236

118

139

2.3

2.4

132

89

44

29

20

20

1.4

G

(mg/dL)

5)

29.8

29

Creatinine

C

5.5

5.9

1.1

0.41

0.38

0.08

8.9

8.4

0.94

0.87

0.46

0.05

Group 2: diabetes (II = 5) Mean SEM

Group 3: renal disease (II = 6) Mean SEM

31.4

31.4

509

230

246

217

161

146

9.2

8.7

5.3

0.8

0.7

128

62

22

23

47

35

1.4

1.1

2

29.5

29.5

936

409t

307

247’

125

127

8.3

7.7

2.9

0.9

0.9

234

87

31

15

19

15

0.7

0.5

0.8

All subjects (n = 16) Mean SEM

Abbreviations: C, control; G, gemfibrozil. *P < ,005. tP < ,001.

a prescribed isocaloric balanced diet (35% fat, 50% carbohydrate, 15% protein) for 2 days before the study. The patients then reported fasting to the Cedars-Sinai outpatient clinic, where blood was drawn for determination of lipids, routine blood chemistries, glycosylated hemoglobin, and thyroid function tests. Fat and muscle biopsies were then performed under local anesthesia. For the fat biopsy, a 3- to 4-cm incision in the lower abdominal wall was used to remove 5 to 10 g fat,16 and for muscle, a needle muscle biopsy removed approximately 300 mg muscle from the quadriceps femoris (vastus lateralis). The patient was then placed on gemfibrozil 600 mg twice a day. Patients receiving hemodialysis or peritoneal dialysis received 300 mg/d gemfibrozil. After receiving gemfibrozil for 6 weeks, the patient again consumed the same isocaloric diet for 2 days, fasting blood was obtained for the above tests, and fat and muscle biopsies were again performed. Thus each patient demonstrated a decrease of serum triglyceride levels, but was unchanged with regard to weight and nutritional state. Together, 17 patients were enrolled in the study, but one patient refused the second biopsies, and therefore this report includes the 16 patients with paired biopsy data. month and consumed

previously.16 LPL activity was expressed as both nanoequivalents of FFA released per minute per lo6 cells and nanoequivalents of FFA released per minute per gram for adipose tissue, and as nanoequivalents of FFA released per minute per gram for muscle. For LPL immunoreactive mass, the identical procedure was performed on a separate piece of fat or muscle, except all buffers and solutions contained the protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 1 mmol/L EGTA). LPL immunoreactive mass was determined by ELISA as described previously.17 The sandwich-type ELISA uses affinity-purified chicken anti-bovine LPL antibodies as a capture antibody; this antibody immunoprecipitates a single band from adipose tissue samples. l7 Following addition of sample or bovine LPL standard, LPL level was measured by the addition of biotinylated affinity-purified anti-LPL antibody, followed by streptavidin-peroxidase. The concentration of LPL in the samples was then calculated using the standard curve for bovine LPL, and was expressed as nanograms per lo6 cells or nanograms per gram for adipose tissue, and as nanograms per gram for muscle. Adipose tissue cell number was determined according to the method of DiGirolamo et al.18 described

LPL Activity and Mass Most of the fat was frozen in liquid nitrogen for subsequent RNA extraction. Frozen samples from pre- and post-gemfibrozil treatment were extracted and processed for Northern and slot blotting together to minimize experimental variation. Another piece of fat was placed into phosphate-buffered saline (PBS) for measurement of LPL activity, cell size, and LPL immunoreactive mass. Samples for LPL immunoreactive mass were prepared on the day of the biopsy, but stored at -20°C prior to enzyme-linked immunosorbent assay (ELISA), such that the pre- and postgemfibrozil samples were assayed together. The muscle tissue was placed into PBS and assayed for LPL activity and mass. Insufficient muscle was obtained for quantitative RNA analysis. Two components of the adipose tissue and muscle were studied, heparin-releasable (HR) LPL and residual extractable (EXT) LPL, as described previously. I6 For LPL activity, the tissue was weighed, rinsed free of blood, minced into smaller pieces, and then incubated in PBS containing 13 WglmL heparin at 37°C for 30 minutes; the buffer containing released LPL was removed and assayed. The tissue was then washed twice in PBS, homogenized in EXT solution (containing detergent and heparin), and assayed as

LPL Synthetic Rate To assess LPL synthesis, pieces of adipose tissue were pulselabeled with 35S-methionine and LPL was immunoprecipitated as described previously.‘g In brief, the adipose tissue was minced and then incubated at 37°C for 30 minutes in medium 199 that was deficient in methionine, followed by an additional incubation for 30 minutes in the same medium to which 50 &i 35S-methionine had been added. The tissue was then homogenized in detergent, and the LPL was immunoprecipitated and analyzed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were loaded with equal quantities of total trichloroacetic acidprecipitable counts to control for any nonspecific effect on total protein synthesis or changes in 35S-methionine incorporation. LPL r&WA Levels The human LPL cDNA probe was labeled to a high specific activity using the “random-priming” method of Feinberg and Vogelstein.” RNA was extracted from adipose tissue using the guanidinium-phenol-chloroform method of Chomczynski and Sac-

1488

SIMSOLO, ONG, AND KERN

chi.21 Northern blots were performed as described previously.ihJy To standardize RNA levels for Northern analyses, the same amount of total RNA from the “before” and “after” treatment specimens was loaded onto the gels, and the equal loading was confirmed by staining the gel with ethidium bromide. To further standardize the quantitation of LPL mRNA, the blots were probed with a radioactive y-actin cDNA probe.22 Images from the Northern blots were quantified by densitometty using a Soft Laser Scanning Densitometer SLR-2D/lD (Zeineh, Fullerton, CA). Because the ratio of actin mRNA to total cellular RNA usually

remains constant, actin cDNA hybridization permitted accurate comparisons between samples.

0.0

HR

EXT

montrol

HR

~

IGemfibrozil

StatisticalAnalysis Results are expressed as the mean ? standard error of the mean. Data were analyzed by the nonparametric Wilcoxon signed rank test for paired samples and by linear regression and correlation analysis.

RESULTS Characteristics of the patients who participated in the study are shown in Table 1. Five patients had hypertriglyceridemia with no evidence of diabetes or renal insufficiency. Another five patients had hypertriglyceridemia along with a history of diabetes. Although glycosylated hemoglobin levels were elevated in all of these patients, two were under fairly good control by diet alone (glycosylated hemoglobin, <7.5) and were not using any diabetes medications. Six patients with hypertriglyceridemia had concurrent renal insufficiency, and four of these patients also had diabetes. Patients generally responded to treatment with gemfibrozil with’the expected decrease in triglyceride levels (P < .OOl). In some cases, blood HDL and low-density lipoprotein (LDL) levels could not be reliably measured because of the degree of hyperlipidemia. In those patients in whom reliable HDL levels were obtained (n = 11) HDL levels increased from 38 mg/dL to 44 mg/dL (P < .05). There was also a significant decrease in total cholesterol levels (P < .005). Gemfibrozil was well tolerated by the patients, and there were no adverse reactions. Data on adipose tissue LPL activity are shown in Fig 1A. Treatment of the patients with gemfibrozil for 6 weeks had no significant effect on adipose LPL activity in either the HR or EXT fraction when expressed as nEq FFA/min/lOh cells (HR, 0.177 2 0.033 and 0.135 ? 0.028, P = .28; EXT, 0.237 * 0.051 and 0.224 + 0.039, P = .23 [before and after gemfibrozil, respectively]) or as nEq FFA/min/g (HR. 0.605 + 0.127 and 0.507 ? 0.117, P = .31; EXT, 0.735 ? 0.215 and 0.857 + 0.161, P = .21 [before and after gemfibrozil, respectively]). In addition, there was no change in LPL immunoreactive mass (HR, 1.685 f 0.337 and 2.319+ 1.093 ng/106 cells [P = .80] or 6.636 2 1.526 and 9.856 2 5.104 rig/g [P = .81]; EXT, 9.4 f 1.838 and 8.797 +- 1.867 ng/106 cells [P = .60] or 36.63 ? 7.43 and 32.86 + 5.77 rig/g [P = .68] before and after treatment, respectively; Fig 1B). The response of adipose LPL to gemfibrozil was also analyzed within each group of patients. There was also no significant change in LPL activity in any of the individual patient groups (data not shown). Correlation analysis was performed between LPL activity levels

50

B

1

d---IL 40

30 3 cn 20 2 10

0

HR

EXT

HR

EXT

Fig 1. Effect of gemfibrozil on LPL activity and mass in adipose tissue. LPL activity (A) and LPL immunoreactive mass (B) were measured in the HR and EXT compartments of adipose tissue. The three experimental groups were pooled together, and results are shown as the mean f: SEM of the 16 subjects studied.

and both HDL and triglycerides, but no significant correlation was found. To further assess LPL gene expression in response to gemfibrozil, adipose tissue from nine patients was pulselabeled with ?S-methionine and immunoprecipitated to assess the protein synthetic rate of LPL in response to gemfibrozil. As shown by the representative gel in Fig 2A, there was no change in LPL synthesis following gemfibrozil treatment. To determine the effects of gemfibrozil on LPL mRNA levels, RNA was extracted from the adipose tissue of 10 patients for Northern blotting. Figure 2A shows Northern blots from two representative patients. Each Northern blot was blotted with the cDNA for LPL and the cDNA for the cytoskeletal protein y-actin, which serves as a constitutive probe and controls for gel loading. Overall, there was no significant change in LPL mRNA levels following gemfibrozil treatment. Images from the Northern blots were quantified using densitometry, and the LPL to y-actin ratios from before and after gemfibrozil treatment were compared. As shown in Fig 2B, gemfibrozil had no effect on the LPL to y-actin mRNA ratios. Muscle LPL activity and mass are shown in Fig 3. There was no change in the level of HR LPL in muscle following gemfibrozil treatment (0.382 +- 0.053 and 0.631 + 0.203 nEq/min/g, P = .49). Although the mean level of EXT LPL in muscle increased following gemfibrozil treatment, this change was not statistically significant (0.683 + 0.267 and 1.427 f 0.631 nEq/min/g, P = .37); four patients demonstrated large ( > fourfold) increases in muscle LPL activity,

1489

EFFECT OF GEMFIBROZIL ON LPL

c

G

CGCG I--

I

..

,

e

LPL 3.6 kb

LPL * 66 kDa

- ACTIN 2.1 kb 2

1

3

4

5

6 125

1 .o

100

0.8

T

0.6

0.4

0.2

0.0

IL1 T

m

Control

75

50

25

0

[~_~]Gemfibrozil

Fig 2. Effect of gemflbrozil on LPL synthetic rate and mRNA levels. Adipose tissue LPL was pulse-labeled with 3%methionine for 30 minutes followed by immunoprecipftation and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (lanes 1 and 2). RNA was extracted from the adipose tissue both before and after gemfibrozil treatment, and the extracted total RNA was analyzed by Northern blotting using the 3zP-cDNAs for LPL and y-actin (lanes 3 through 6). (B) Images from Northern blots of 10 patients quantified using densitometry. LPL to yactin ratios were expressed in arbitrary units and as a percentage of the LPL to yactin ratio during the control period. (C)Control (before gemfibrozil treatment); (G) after gemfibrozil treatment.

whereas most patients demonstrated no change or a decrease in LPL activity. The four patients who demonstrated increases in muscle LPL activity were from all treatment groups and were not clinically distinguishable from the other patients. There was no change in LPL immunoreactive mass in either HR or EXT components (HR, 11.01 f 5.59 and 17.58 & 7.65 rig/g,, P = .19; EXT, 39.01 2 18.2 and 41.41 2 16.71 rig/g,, P = .43). DISCUSSION

Gemfibrozil is a potent triglyceride-lowering drug that has been used extensively in the treatment of hypertriglyceridemia. Any drug that decreases plasma triglyceride levels would be expected to do so either by decreasing VLDL triglyceride output by the liver or by increasing triglyceride-rich lipoprotein removal. Triglyceride-rich lipoproteins are catabolized through the action of LPL, which is found predominantly in adipose tissue and muscle. Therefore, this study was designed to examine the mechanism of gemfibrozil action in hypertriglyceridemic humans by examining the effect of gemfibrozil treatment on LPL expression in the tissues of LPL synthesis. In this study, patients were treated with gemfibrozil for 6 weeks, at which time plasma triglyceride levels were lower and patients were unchanged with regard to weight, diet,

other medications, or other possible confounding variables. There was no change in LPL activity, mass, synthetic rate, or mRNA levels in fat, and no significant change in muscle LPL activity or mass. Several patients demonstrated increases in muscle LPL activity, but these changes were not seen consistently and were not statistically significant. Although muscle LPL activity did not change in the group, the sporadic increases in several patients raise the possibility that there may be much interindividual variation. It is possible that changes in LPL following gemfibrozil treatment represent a variable that determines the magnitude of the triglyceride-lowering response to the drug. Perhaps gemfibrozil decreases circulating triglyceride levels primarily by decreasing VLDL production, coupled with varying degrees of increased VLDL removal. In some patients, the increased removal may be an important mechanism. It would be useful to determine the reason for this heterogeneity of response to further the development of drugs that regulate lipid metabolism. A number of previous studies have examined the effects of fibrates on LPL in humans. An important mechanism for decreasing circulating triglyceride levels by gemfibrozil and clofibrate is a decrease in VLDL output.6*9Jn In addition, a number of studies have described either increased triglyceride-rich lipoprotein removal or increased postheparin

1 EXT

n

Control

/I

L

Gemfibrozil

B L HR

EXT

Fig 3. Effect of gemfibroxil on LPL activity and mass in muscle. LPL activity (A) and LPL immunoreactive mass (B) were measured in the HR and EXT compartments of muscle tissue. The three experimental groups were pooled together, and results are expressed as the mean + SEM of 10 subjects studied.

1490

SIMSOLO, ONG, AND KERN

plasma LPL levels after treatment with gemfibrozil.h~1i-‘5 Although most studies have measured LPL levels in postheparin plasma, adipose tissue LPL levels have been shown to increase in response to c1ofibrate.s However, studies on the effect of gemfibrozil on LPL have not been consistent. Gnasso et alz3 found no increase in postheparin plasma LPL activity in normolipidemic subjects during the course of 12 weeks of therapy with gemfibrozil. In the study chylomicron by Weintraub et al,” retinyl palmitate-labeled clearance was studied in patients with type IV hyperlipidemia. Although gemfibrozil therapy produced an increase in clearance of retinyl palmitate-labeled chylomicrons and chylomicron remnants, there was no increase in postheparin plasma LPL activity. The reason for this apparent paradox-an increase in chylomicron clearance in the absence of a change in LPL-is not clear. How does one reconcile these data with those of other studies that showed increases in postheparin plasma LPL activity following gemfibrozil treatment? Most other studies did not directly examine adipose tissue and muscle LPL. but measured LPL activity in postheparin plasma. It is not exactly clear what relationship exists between postheparin plasma LPL and tissue LPL. Postheparin plasma LPL is a mixture of LPL from both adipose tissue and muscle that is displaced by heparin from glycosaminogiycan binding sites on endothelial cells. Following release from endothelial cells, postheparin plasma LPL circulates bound to lipoproteins in the intermediate-density lipoprotein and LDL density class.24 There are several possible explanations for an increase in postheparin plasma LPL activity in the absence of an increase in tissue LPL activity. Gemfibrozil

may stimulate translocation from the cell or interstitial space to the endothelium and not affect LPL production. With the recent identification of an LPL-binding protein on endothelial cells,25 the capacity for LPL binding to the endothehum may be subject to regulation. On the other hand, gemfibrozil may function to stabilize the heparinreleased LPL protein in plasma. Because LPL in postheparin plasma is associated with intermediate-density lipoprotein,24 there is likely an ongoing triglyceride hydrolysis while the heparin-released LPL circulates through plasma. In hypertriglyceridemic plasma. this circulating lipoproteinbound LPL may become partially exhausted following longstanding hydrolysis of large, triglyceride-rich VLDL particles. When the number and size of triglyceride-rich lipoprotein particles is decreased by gemfibrozil, a more active form of LPL may ensue because of the decreased availability of triglyceride substrate, thus producing a limited need for LPL hydrolysis and subsequent inactivation. In summary, we studied LPL gene expression in 16 patients with hypertriglyceridemia who were treated with gemfibrozil. Although serum triglyceride levels were decreased by gemfibrozil therapy, there was no significant change in LPL expression. These data suggest that the predominant mechanism for the decrease of triglyceride levels by gemfibrozil is inhibition of VLDL production. ACKNOWLEDGMENT We wish to thank Dr John Goers for the anti-LPL antibodies. and Mehrnoosh Ghiam and Bahman Saffari for technical assistance. We also wish to thank Dr Jan Bonalsky Warner Lambert for his helpful comments.

of Parke-Davis/

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1491

EFFECT OF GEMFIBROZIL ON LPL

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