Lipolytic Enzymes as markers of induction and differentiation

coO9-9120/87 ml0 + .oo Copyright 0 1937 The Canadian Society of Clinical Chemists.

Clin Biochem, Vol. 20, pp. 406-413, 1987 Printed in Canada. All rights recwved.

L,ipoI’ytic Enzymes as Markers of induction and Differentiation DAVID M. GOLDBERG and JOEL G. PARKES Department of Biochemistry, The Hospital for Sick Children and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada Factors leading to microsomal enzyme induction are associated with hypertriglyceridemia in man. Phenobarbital (PB) increases hepatic synthesis of triglyceride but lowers its serum concentration in rats due to increased postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL); these changes are accompanied by increased activity of these lipolytic enzymes in adipose tissue and liver. The present work explores the cellular mechanisms whereby PB increases the tissue content of these enzymes, using primary cultures of rat liver hepatocytes and a continuous cell line of mouse ffbroblasts (preadipocytes) that undergo differentiation into mature fat cells. Secretion and synthesis of HTGL in primary rat hepatocytes increased 50% with insulin; when PB was added with insulin, activity was enhanced an additional 50%. By contrast, insulin inhibited HTGL secretion from the well differentiated rat hepatoma cell line, FU-55,C8, and this inhibition was partly overcome by PB. These results suggest that different control mechanisms govern the synthesis and secretion of HTGL in normal rat liver cells and hepatoma. In cultured pre-adipocytes (3T&F442A) insulin promoted differentiation when added to confluent cultures. PB (0.5 mM) resulted in marked enhancement of conversion of adipocytes characterized by a two- to threefold increase in extracellular LPL and a 1O-foldincrease in intracellular enzyme. These results suggest that PB promotes conversion of uncommitted cells into pre-adipocytes at an early stage in the differentiation of adipose tissue. KEY WORDS: lipoprotein lipase; hepatic triglyceride lipase; triglyceride; phenobarbital; insulin; enzyme secretion; hepatoma; adipose conversion; hepatocyte culture.

Introduction CLINICALSTUDIESINHYPERTRIGLYCERIDEMIA

T

he investigations described in this report were stimulated by a series of clinical studies which demonstrated a significant correlation between the serum triglyceride (TG) concentration and the serum activity of the enzyme gamma-glutamyl transferase (GGT; EC 2.3.2.2) in the following conditions: (a) untreated patients with hypertension presented to a high blood pressure clinic for the first time (1);

Correspondence: Dr. D. M. Goldberg, Department of Biochemistry, The Hospital For Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Manuscript received October 24, 1986; revised June 12, 1987; accepted June 16, 1987. CLINICAL BIOCHEMISTRY,

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20, DECEMBER

1987

(b) patients with diabetes mellitus (2); and (c) healthy females taking oral contraceptives

(3).

Since serum GGT activity is an index of hepatic microsomal enzyme induction (4), and since most of the circulating TG is synthesized by enzymes located in microsomes in the liver (51, we reasoned that enzymeinducing agents might contribute to hypertriglyceridemia by switching on the synthesis of TG-synthesizing enzymes in liver microsomes (6). PHENOBARBITALANDTRIGLYCERIDEMETABOLISM

Effect on triglyceride

synthesis

The hypothesis that hypertriglyceridemia and microsomal enzyme induction are related was tested in animals injected with phenobarbital (PB) under conditions that generated the maximum increase in cytochrome P-450-dependent microsomal enzymes (7). We were able to show that under these circumstances, PB induced increased activities of hepatic TG-synthesizing enzymes, as well as increased hepatic TG content in rabbits, guinea pigs, and rats (8-11). We were also able to show that the increased activity of cytochrome P450-dependent enzymes and hepatic GGT activity correlated closely with: (a) an increase in TG-synthesizing enzymes such as phosphatidate phosphohydrolase (PPH; EC 3.1.3.4) and diacylglycerol acyltransferase (DGAT; EC 2.3.1.20); (b) an increase in the in vitro incorporation of precursors such as glycerol-3-phosphate into TG; and (cl an increased content of TG in the liver. It was expected that increased hepatic synthesis of TG and increased hepatic content of TG would lead to a significant increase in the fasting concentration of circulating TG in the blood serum, and this indeed proved to be so in the rabbit (9) and guinea pig (101, but in the rat, not only was serum TG concentration not increased; it actually fell significantly during PB treatment (8,111. There were two possible explanations for this unexpected finding in the rat: PB must either decrease the rate of TG secretion by the liver, or it must increase the rate of degradation and clearance of TG in the rat circulation. Possible changes of hepatic TG-secretion rate in PB-treated rats were ruled out by mea405

GOLDBERGAND PARKES suring this directly by means of the Triton WR 1339 technique ( 11). Phenobarbital

and postheparin plasma lipolysis

The effect of PB upon TG-clearing enzymes of rat plasma was also studied and it was found that PB dramatically increased the plasma postheparin lipolytic activity in this animal (12). Using protamine sulfate and 1 M NaCl to inhibit lipoprotein lipase (LPL; EC 3.1.1.34) and sodium dodecyl sulphate to inhibit hepatic triacylglycerol lipase (HTGL; EC 3.1.1.31, it was shown that both LPL and HTGL were significantly increased in the serum of PB-treated rats. Circulating LPL arises predominantly from skeletal muscle, cardiac muscle, and adipose tissue (13). We were able to show that PB did not increase the LPL activity of skeletal muscle or cardiac muscle, but it did significantly increase the LPL content of adipose tissue (12). Obviously, with HTGL, there is only one possible tissue source-the liver-and we demonstrated that PB also increased the HTGL content of liver homogenates and microsomal fractions (12). The fact that PB enhanced HTGL activity in the liver came as no surprise, but it was very surprising to discover that it also increased LPL activity in adipose tissue, since this tissue is considered to be totally inert so far as drugs are concerned, which is why fat soluble drugs may persist in adipose tissue long after they have been metabolized and cleared from other tissues (14). Indeed, our own unpublished investigations failed to detect any drug-metabolizing enzymes or cytochrome P-450 in microsomal preparations of adipose tissue from rats manifesting high levels of hepatic microsomal cytochrome P-450. We could not characterize the effect of PB upon adipose tissue in whole animals because we were unable to distinguish between a direct effect of the drug, and an effect which was simply modulated by other hormonal regulators of tissue metabolism whose concentrations might be modified by PB. We therefore decided to explore the interaction between PB and adipocytes in cell culture systems. Similar reasoning led us to study the effect of PB upon rat hepatocytes grown in culture. Studies with liver cells HEPATOCYTECULTURE Primary hepatocytes from rat liver were isolated by perfusing the portal vein first with EGTA, and subsequently with collagenase before dissociating the hepatocytes in Liebovitz medium (15,161. The cells collected by brief centrifugation were more than 90% viable by the criterion of Trypan Blue exclusion and were seeded onto collagen-coated culture plates. They were divided into four groups and maintained for three days in (a) basal growth medium alone; (b) basal medium supplemented by insulin alone; (c) basal medium plus PB alone; or (d) basal medium with PB and insulin together. To measure the extracellular HTGL, we first removed the growth medium, washed the monolayer, 406

and then incubated it for 1 h with a buffered salt solution containing heparin. At the end of this incubation period, the heparin solution was removed and its HTGL activity was taken to be that of the extracellular enzyme. The cells were then scraped from the culture plates and ruptured by sonication. The HTGL activity of the sonicated cells was taken to represent the intracellular enzyme. PHENOBARBITAL AND HTGL SECRETION Hepatocytes, which had been grown in the presence of insulin for three days, showed a significant increase in both intracellular and extracellular HTGL per plate compared with cells that had been grown in basal medium alone (Table 1, top). The increases in intracellular and extracellular enzymes were proportionally the same, and this suggested that insulin brought about a balanced increase in both synthesis and secretion. When PB was added to the medium along with insulin, there was a further significant increase in extracellular HTGL, but this was accompanied by a significant reduction in intracellular enzyme by comparison with cells treated with insulin alone. Since insulin and PB stimulate protein synthesis by the liver, the activity of HTGL in relation to hepatocyte protein content was also measured. The HTGL activity per mg protein in both the intracellular and extracellular fractions was increased by insulin over that of cells grown in the basal medium, but these increases were not statistically significant (Table 1, centre). On the other hand, the addition of PB along with insulin significantly increased the extracellular HTGL specific activity and significantly reduced its intracellular specific activity compared with those parameters in cells grown in the presence of insulin alone (Table 1, centre). The data so far suggest that insulin stimulates the synthesis and secretion of HTGL, and that when PB is present, there is a further stimulation of HTGL secretion accompanied by a decrease in intracellular pools of the enzyme. This is best appreciated by expressing the data as a percentage of its distribution between intracellular and extracellular pools. As shown in Table 1 (bottom), the intracellular HTGL as a percentage of the total activity of this enzyme in the presence of insulin was not significantly different from that of cells grown in basal medium alone, but when PB was added along with insulin, a significant reduction took place in the percentage of total HTGL activity present intracellularly. At a fixed concentration of insulin, PB increased the extracellular and total HTGL activity up to a concentration of 0.5 mM (Figure 11, but beyond that concentration, PB reduced HTGL activity, probably due to a toxic effect of the drug upon the cells. When PB was maintained at 0.5 mM and insulin was added in increasing amounts, total HTGL activity increased and reached a plateau around 10 mU/mL insulin. The effect of insulin was enhanced by PB, and this enhancement was more marked at higher insulin concentrations (Figure 2). CLINICAL BIOCHEMISTRY,VOLUME 20, DECEMBER 1967

LIPOLYTIC ENZYMES TABLE 1 Effectof InsulinandPhenobarbital UponTriglycerideLipase Activityin Rat Hepatocytes TriglycerideLipaseActivity

Culture conditions

Intracellular

Extracellular

Nanomoleoleic acid/plate Basalmedium

29.7 -c 11.6 (14) ‘31.3 + 18.7 (25) b26.0* 14.9 (25)

Insulin(10 mU/mL) Insulin + 0.5 mM Phenobarbital

136 * 97.5 (14) “202 2 75.0 (37) b241? 102 (371

Nanomoleoleic acid/mgprotein Basalmedium

10.3 + 3.3 (14) ‘14.0 + 7.3 (24) b12.1rt 6.7 (24)

Insulin(10 mU/mL) Insulin + 0.5 mM Phenobarbital

85.2 + 28.3 (14) ‘113 * 64.2 (27) b131t 51.8 (27)

Percentof total activitv Basalmedium

17.7 f 4.5 (14) Cl58 2 5.3 (22) b12.55 5.6 (221

Insulin(10 mU/mL) Insulin + 0.5 mM Phenobarbital

82.3 t 7.0 (14) ‘84.2 2 5.3 (22) b87.5* 5.6 (22)

In preliminary experiments with these highly differentiated cells, we demonstrated the presence of a lipase that was not inhibited by high salt concentration or stimulated by serum, and which was released by adding heparin to intact cells, suggesting that it was similar or identical to HTGL found in rat liver and hepatocyte cultures. Surprisingly, this enzyme was completely unresponsive to insulin (Figure 2) and no change in extracellular or intracellular HTGL was observed (Figure 3). We examined the effect of insulin upon amino acid uptake by these cells, since it is a well established function of insulin that requires the presence of insulin receptors on the cell surface (18). No significant effect of insulin upon amino acid uptake by C-8 hepatoma cells was detected, suggesting that either these cells lack functional insulin receptors, or that their postreceptor function is impaired. In fact, insulin actually inhibited HTGL secretion from the hepatoma cells, and this inhibition was partially overcome by PB. Although these results are very preliminary, they suggest that different control mechanisms govern the synthesis and secretion of HTGL in normal rat liver cells and in hepatoma; in the hepatoma cells, there appears to be the loss of an important regulatory mechanism associated with insulin receptors. This is consistent with the varied responses to insulin and other hormones of rat hepatocytes and hepatoma cell lines reported by Kelley et al. (19).

Statistical significance using t test as follows: ‘p < 0.01 (Basal vs Insulin); “p < 0.01 (Insulin vs PB, paired data); and 'p < 0.05 (Basal vs Insulin). Data shown as mean 2 SD.

Studies with cultured adipocytes

HTGL SECRETION IN HEPATOMA CELL LINE

We used a line of mouse fibroblasts (3T3-F442A), which behave as pre-adipocytes, differentiating into

Because primary hepatocyte cultures are limited by their rather short viability, we decided to study a rat hepatoma cell line (FU-5-5,C-81 that grows indefinitely and is useful in examining hepatocyte functions since these are largely preserved in this cell line (17).

100 I

50 . .

m -+

0 (>-o----h 0

0.5

1.o

Intracellular I\ Q 1.5

2.0

Phenobarbital Concentration (mM) Figure 1-Hepatic triglyceride lipaae activity versus phenobarbital concentration in primary rat hepatocytes. CLINICAL BIOCHEMISTRY, VOLUME 20, DECEMBER 1987

CULTURE OF ADIPOCYTES

Rat hepaiocytes, no phenobarbfial Rat hapatoqles.

phenobarbital 0.5 mM

d1 1

5

Insulin Concentration

40

(mU/ml)

Figure 2-Hepatic triglyceride lipaae activity in primary rat hepatocytes and rat hepatoma (C-8) grown with various insulin concentrations.

407

GOLDBERGAND PARKES

r-

5

~~

.

7

6

6

9

Time after Addition (days)

5

7

6

8

9

Time after Addition (days)

Figure 3-The effect of growth conditions upon: (A) extracellular and (B) intracellular hepatic triglyceride lipase in C-8 hepatoma. Shaded circle denotes basal growth medium; open circle denotes insulin (10 mU/mL).

mature adipocytes under appropriate culture conditions. These cells acquire many of the features of mature adipocytes in uiuo (20) and are useful for examining drug-adipose tissue interactions. We monitored two characteristics of adipocyte conversion: the ability to synthesize and secrete LPL; and the acquisition of intracellular TG. The cells were grown for about five to seven days until the monolayers were confluent. When insulin is added, a high proportion of these cells differentiate into mature adipocytes. This differentiation was characterized by the development of easily visible fatty colonies that could be seen on phase-contrast microscopy, or after staining the cultures with Oil-red-O (Figure 4). If culture is continued in the basal growth medium, very few of the cells differentiate into adipocytes. As in the previous experiments with hepatocytes, we measured both intracellular and extracellular LPL. The latter was released by incubating monolayer cultures with heparin; the enzyme remaining within the cells after removing the heparin solution and sonicating was taken to represent the intracellular LPL (21). EFFECTOFPBONADWXYTE

DIFFERENTIATION

The addition of insulin brought about an increase in the TG content of the cultured cells that was most 408

Figure PDifferentiated Oil-red-O.

3T3-F442A adipocytes stained with

marked by the 15th day of incubation (Figure 5). The addition of PB along with insulin enhanced TG production at times earlier than the 15th day, suggesting that PB stimulated adipocyte differentiation beyond that accomplished by insulin alone. PB by itself had no effect but appeared to act synergistically with insulin. The protein content of these cultures was also stimulated by insulin, starting around the eighth or ninth day and reaching a maximum by day 15 which was the last day of the experiment (Figure 6). Once more, PB together with insulin greatly enhanced protein content, starting around the fifth day and increasing somewhat unevenly up to the last day of observation. PB by itself did not have much effect. These results indicated that treatment with insulin or with insulin plus PB, increased protein content twoto threefold and TG levels some 20-fold in 3T3 preadipocytes. Differentiation did not occur in cells grown in basal medium or with PB alone, and consequently there was no noticeable increase in their protein or TG content. The total LPL activity was increased by insulin, but in the presence of PB, the increase was even more dramatic, starting around days 4 to 5, increasing to a peak at day 7 and then showing a decline (Figure 7). Thus, cells undergoing differentiation acquire LPL activity, and this enzyme emerges several days earlier CLINICAL BIOCHEMISTRY,VOLUME 20,DECEMBER

1987

LIPOLYTIC ENZYMES

63

Growth medium alone

w

Insulin

0

Phenobarbital

n

Insulin + phenobarbital

.

2

4

5

6

7

8

9

-I

12 13 14 15

Days of Induction Figure

&Triglyceride content in differentiating 3T3-F442A cells under varying culture conditions.

than the increase in protein or TG (22), and is therefore a much earlier marker of differentiation. This indeed is what one would expect, since LPL hydrolyses extracellular circulating TG in VLDL and chylomicrons to

produce free fatty acids which are taken up by the adipocyte for the synthesis of its own stored TG (23). EFFECTOFPB ONLPL POOLS The extracellular LPL content of pre-adipocytes cultured in the presence of PB and insulin was much greater than that of cells grown in the presence of insulin alone (Figure 8), and this enhancement was maximal by the sixth day of culture. The intracellular LPL was dramatically increased by PB in the presence of insulin compared with cells grown in insulin alone, and in this instance the increase was maximal by the ninth day (Figure 9). Comparing the behaviour of total LPL activity with the behaviour of the extracellular (Figure

3.5 3.0 mm g

2.5 ..

3

2.0 .*

=

1.5 ..

Growth medium alone

m

Insulin

0

Phenobarbital

EFFECTOF PB UPONDAILY LPL SECRETIONRATE

To determine the effect of PB upon the daily rate of LPL secretion as well as on the time required to reach the maximum daily output, 3T3 cells were grown to confluence and their growth was continued in media containing insulin alone or insulin plus PB. After four days, the medium was removed and the cells were incubated with heparin for 60 min to measure the extracellular LPL. Growth of the cells in the same media was continued, and each day, until the 11th day of the

Insulin + phenobarbital

2 Figure 6-Protein

69

8) and intracellular (Figure 9) LPL independently, it is evident that around the time of maximal LPL production, the extracellular activity accounts for the bulk of total LPL. However, at later times, the intracellular activity becomes more significant. Thus, the extracellular rather than the intracellular enzyme is the earliest marker of differentiation in these cultures.

4

5

6

7 8 9 12 13 Days of Induction

1

14 15

content of differentiating 3T3-F442A cells under varying culture conditions.

CLINICAL BIOCHEMISTRY,VOLUME 20, DECEMBER 1987

409

GOLDBERG AND PARKES

7000 0 Z$

5000

.g.g 3&Z :gg

4000 6000

69

Growth medium alone

I 0 I

insulin Phenobarbital insulin + Phenobarbital

I

3000 2000 1000 0

u 2

4

5

6

1 8

7

Days of Induction Figure 7-Total

lipoprotein

lipase activity

in differentiating

experiment, heparin incubation was repeated and the extracellular LPL measured. As shown in Figure 10, cells treated with PB demonstrated a greater rate of LPL secretion at all times. The maximal rate of LPL secretion occured on the eighth day. On the 11th day, when the experiment was terminated, the intracellular LPL was measured, and as expected, this was greatly enhanced in the PB-treated cells, represented by the hatched column, compared with cells treated with insulin alone as indicated by the stippled column in Figure 10.

3T3-F442A

cells under varying culture conditions.

was measured at intervals throughout this period (Figure 11). Compared with those cells that did not receive PB and that are represented by the solid circles, the highest activity was present in those cells indicated by the open circles and to which PB had been added at day 0 (Figure 11). Cells indicated by the open squares in which PB was started on day 2 had lower activity, and this was still lower in the cells indicated by the solid squares and to which PB treatment was only commenced on day 4. Thus, the magnitude of the increase in LPL was critically dependent upon the timing of PB addition.

!hMING OF PB ADDITION STUDIES ON 3T3-Ll

To determine whether PB was able to stimulate the expression of differentiation after it had been initiated by insulin, 3T3 cells were grown to confluence, after which they were treated with insulin. PB was then added at different days after insulin addition. Growth was continued for a further 14 days, and LPL activity

PREADIPOCYTES

Another continuous line of 3T3 fibroblasts can also differentiate into adipocytes when grown in the pres-

6OOOr m

JL

24567

8

9

Insulin + phenobarbital

12 13 14 15

Days of Induction Figure 6-Extracellular lipoprotein lipase activity in differentiating 3T3-F442A cells with insulin in the presence and absence of phenobarbital. 410

245678 Days of Induction Figure 9-Intracellular lipoprotein lipase activity in differentiating 3T3-F442A cells with insulin in the presence and absence of phenobarbital. CLINICAL BIOCHEMISTRY, VOLUME 20, DECEMBER 1987

LIPOLYTIC ENZYMES

4-7

7-8

m

Extracellular, Insulin

0

Extracellular, Insulin + PB

m

Intracellular, Insulin

m

Intracellular, Insulin + P8

8-9

Time After Induction (days) Figure lo-Daily

output of lipoprotein lipase in differentiating 3T3-F4542A

ence of insulin. When PB was added together with insulin, differentiation was augmented and with it total LPL activity (Figure 121,although the increase was less dramatic than that which occurred with the F442A cells. This cell line also undergoes differentiation to mature adipocytes when stimulated by a combination of dexamethasone and methyl-isobutyl xanthine. PB, when added to dexamethasone plus methyl-isobutyl xanthine, enhanced differentiation and with it LPL secretion and intracellular content (Figure 13). Even when cultures were stimulated by insulin together with dexamethasone and methyl-isobutyl xanthine, the addition of PB brought about enhancement of extracellular and intracellular LPL (Figure 13).

l

Insulin, ~2 phenobarbital

0

Insulin+ phembatbiial (day 0) Insulin+ phenobarbital (day 2)

1*

n

Insulin+ phenobatbiial (day 4)

cells.

INTERPRETATION 0~ DATA The following model of adipocyte differentiation has been proposed (24,251. First, there is a commitment of stem cells or adipoblasts to become pre-adipocytes; next, there is proliferation of pre-adipocyte clones or foci; next, there is initiation of differentiation; and finally, expression of the differentiation program occurs. In cultures, committed cells proliferate among noncommitted cells to form clones of pre-adipocytes that are susceptible to differentiation. These clones vary in size, depending upon the number of cells present initially. Upon stimulation with insulin or other promoters of differentiation, foci emerge as the pre-adipocytes begin to acquire the attributes of mature adipocytes by synthesizing a new complement of enzymes and accumulating TG (Figure 14). The best interpretation of our data is that the number of these foci is increased when 3T3 cells are exposed to PB, provided that insulin is also present. Since the time required to reach maximum LPL production is the same for cells treated with insulin in the presence or absence of PB, the drug is probably not a specific stimulus for

1

4

5

6

7

8

9

Time after Induction

10

11

12

13

14

6

8

10

12

14

16

(days)

Figure 11-The effect of phenobarbital upon lipoprotein lipaae activity when added to 3T3-F442A cells at various stages of differentiation. CLINICAL BIOCHEMISTRY, VOLUME 20, DECEMBER

1987

Days of Induction Figure 12-Effect of insulin and phenobarbital upon total lipoprotein lipase activity of 3T3-Ll pre-adipocytes. 411

GOLDBERG AND PARKES

0

insulin/dex/mix

m

insulin/dex/mix/pb

q q

d&mix dex/mix/pb insulin

n

Extracellular

ins~~lii + PB

Intracellular

Figure 13-Effect of phenobarbital upon induction promoted by dexamethasone, methyl-isobutyl xanthine (mix), and insulin in 3T3-Ll preadipocytes.

the final stages in adipocyte conversion that modulate the expression of the differentiated phenotype. Thus PB does not seem to affect the fourth and final stage of differentiation. Because the time course of increased intracellular LPL, protein, and TG production is the same in the presence of PB as in its absence, the drug does not seem to stimulate the onset of differentiation, that is stage 3 of the model, otherwise these differentiated characteristics would emerge much earlier. If PB were to enhance the proliferation of preadipocytes after

confluence, that is stage 2 of the model, then the size of the foci would be greater in the PB-treated cultures. However, this was not the case, since we observed that the number of foci rather than their size was increased by adding PB. Our data are thus best explained by the proposal that PB modulates the first stage of adipocyte differentiation, that is the commitment of the stem cells or adipoblasts to become pre-adipocytes. Some forms of obesity depend upon hypertrophy of adipocytes, which come to contain excessive amounts of TG, whereas others are due to adipocyte hyperplasia suggesting enhanced commitment of stem cells to differentiation. The studies we have described demonstrate that drugs can modulate the first stage of this process. They also describe an experimental model which can enable the very earliest stages of adipocyte differentiation to be studied, and they characterize extracellular LPL secretion as an early and readily measurable index of differentiation. We believe that these results should make a contribution to the study of obesity which has become a major cause of disease and di&urement in our present era of opulence and self-indulgence. Acknowledgement Thesestudies were supported by grants from the Ontario Heart and Stroke Foundation, and the Medical Research Council of Canada. We thank Mr. R. Hussain and Mr. P. Chan for expert technical assistance.

6 ”

Figure 14-Model

412

I

of pre-adipocyte differentiation.

References 1. Martin PJ, Martin JV, Goldberg DM. Gamma-glutamyl transpeptidase, triglycerides, and enzyme induction. Br Med J 1975; 1: 17-18. 2. Martin JV, Hague RV, Martin PJ, Cullen DR, Goldberg DM. The association between serum triglycerides and gamma-glutamyl transpeptidase activity in diabetes mellitus. Clin Biochem 1976; 9: 208-11. CLINICAL BIOCHEMISTRY, VOLUME 20, DECEMBER 1987

LIPOLYTICENZYMES 3. Martin JV, Martin PJ, Goldberg DM. Enzyme induction as a possible cause of increased serum-triglycerides after oral contraceptives. Luncet 1976; 1: 1107-8. 4. Goldberg DM. The expanding role of microsomal enzyme induction and its implications for clinical chemistry. Clin Chem 1980; 26: 692-9. 5. Hubscher G. Glyceride metabolism. In: Wakil SJ Ed. Lipid metabolism. Pp. 280-370 London and New York: Academic Press, 1970. 6. Goldberg DM, Martin JV, Martin PJ. The association between serum gamma glutamyl-transpeptidase activity and triglyceride concentration in a constellation of clinical situations. In: Griffiths JC Ed. Clinical enzymology. Pp. 187-98, New York: Masson, 1979. 7. Lu AYH, Kuntzman R, Conney AH. The liver microsomal hydroxylation enzyme system. Induction and properties of the functional components. In: Van der Reis L Ed. Frontiers of gastrointestinal research, Vol 2. Pp. 1-31 Basel: S. Karger, 1976. 8. Goldberg DM, Roomi MS, Yu A, Roncari DAK. Effect of phenobarbital on triglyceride metabolism in the rat. In: Coon MJ, Conney AH, Estabrook RW, Gelboin HV, Gillette JR, O’Brien PJ Eds. Microsomes, drug oxidations, and chemical carcinogenesis. Pp. 725-8, New York: Academic Press, 1980. 9. Goldberg DM, Roomi MW, Yu A, Roncari DAK. Effects of phenobarbital upon triacylglycerol metabolism in the rabbit. Biochem J 1980; 192: 165-75. 10. Goldberg DM, Yu A, Roomi MW, Roncari DAK. Effects of phenobarbital upon triacylglycerol metabolism in the guinea pig. Can J Biochem 1981; 59: 48-53. 11. Goldberg DM, Roomi MW, Yu A, Roncari DAK. Triacylglycerol metabolism in the phenobarbital-treated rat. Biochem J 1981; 196: 239-48. 12. Goldberg DM, Roomi MW, Yu A. Modulation by phenobarbital of lipolytic activity in postheparin plasma and tissues of the rat. Can J Biochem 1982; 60: 1077-83. 13. Smith LC, Pownall HJ. Lipoprotein lipase. In: Borgstrom B, Brockman HL Eds. Lipuses Pp. 263-305 Amsterdam: Elsevier Science Publishers BV, 1984. 14. Benet LZ, Sheiner LB. Pharmacokinetics: the dynamics

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of drug absorption, distribution, and elimination. In: Gilman AG, Goodman LS, Rall TW, Murad F Eds. Thepharmucological basis of therapeutics, 7th od Pp. 3-34 New York: MacMillan Publishing Company, 1985. Chan P, Parkes JG, Goldberg DM. Secretion of triglyceride lipase from rat hepatocytes and hepatoma: modulation by insulin and phenobarbital. Proc Can Fed Biol Sot 1986; 29: 221 (Abst). Parkes JG, Chan P, Goldberg DM. Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital. Biochem Cell Bioll986; 64: 1147-52. Widman LE, Chasin LA. Multihormonal induction of azUglobulin in an established rat hepatoma cell line. J Cell Physiol 1982; 112: 316-26. Fehlmann M, Le Cam A, Freychet P. Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport. J Biol Chem 1979; 254: 10431-7. Kelley DS, Becker JE, Potter VR. Effect of insulin, dexamethasone, and glucagon on the amino acid transport ability of four rat hepatoma cell lines and rat hepatocytes in culture. Cancer Res 1978; 38: 4591-600. Kuri-Harcuch W, Green H. Increasing activity of enzymes on pathway of triacylglycerol synthesis during adipose conversion of 3T3 cells. J Biol Chem 1977; 252: 2158-60. Parkes JG, Goldberg DM. Phenobarbital modulates differentiation of 3T3 preadipocytes.Fed Proc 1986; 45: 1579 (Abst.). Ailhaud G. Adipose cell differentiation in culture. Mol Cell Biochem 1982; 49: 17-31. Cryer A. Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Znt J Biochem 1981; 13: 52541. Ailhaud G, Amri E, Cermolacce C, et al. Hormonal requirements for growth and differentiation of 0B17 preadipocyte cells in vitro. Diabete metabolisme 1983; 9: 12533. Parkes JG, Hussain RA, Goldberg DM. Modulation by phenobarbital of the differentiation of 3T3 preadipocytes. Biochem Cell Bioll986; 64: 1141-6.

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