Vol. 7, Part II, pp . 1303-1309, 1968 . Life Sciences Printed in Great Britain.
Pergamon Press
REGULATION OF ADIPOSE TISSUE LIPOPROTEIN LIPASE SYNTHESIS BY INTRACELLULAR FREE FATTY ACID E sko A. Nikkilä and Olavi Pykälistö Third Department of Medicine, University of Helsinki, Helsinki 29, Finland
(Received 19 September 1968 ; in final form 17 October 1968) The lipoprotein lipase (LPL) of adipose tissue is one of the most adaptive animal enzymes and seems to form a promising model for studies of 3 S enzyme regulation. The LPL activity is decreased on fasting ' and on acute exercise
12
as well as in experimental diabetes7'
18
but it is rapidly restored
by feeding glucose to fasted animals or .giving insulin to diabetic rate . Nicotinic acid was recently shown to cause a rapid induction of LPL in fasting rats 11 .
The enzyme has an exceptionally rapid turnover 11, 19 and it is syn-
thesized under suitable conditions in vitro 6 ' l ~.
The ultimate regulatory
devices operating in LPL synthesis are not known, however. Under all the conditions mentioned above the LPL activity is inversely related to the extent of lipolysis of intracellular fat and, therefore, the possibility was raised that a product of lipolysis, e . g . the free fatty acid (FFA), might be a regulating 15 . fa~aor in the synthesis and activity of LPL Some data support this
2~ view 15, and particularly the discovery of a rapid induction of the enzyme by nicotinic acid in vivo but not in vitro strexigthened the hypothesis . In the present communication it will be shown that under all experimental conditions with variable ra.trs of lipolysis or of FFA rc-estcrification the in vitro synthesis of adipose tissue 1~PL is closely relat~" d to
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1304
LIPASE SYNTHESIS
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FFA release being stimulated by low and repressed by high FFA concentrations. Methods All experiments were carried out in vitro with the epididymal adipose tissue of white Wistar strain rats weighing 160 to 180 grams. The animals were fed ad libitum and given 5 per cent glucose as drinking fluid. Although it should have been more natural to study the induction of low in vivo LPL activity, glucose-fed animals were used in preference of fasting ones to avoid depletion of those enzymes and metabolites which maintain favorable energetic conditions for new pratein synthesis. It was also thought that tissue from a fed animal should provide more "pure" conditions for the study of LPL induction when there were no secondary rate-limiting reactions, e, g. of the glucose metabolism as might be the case in tissue taken from fasted rats . Immediately after sacrifice the epididymal fat pads were scissed off and cut into pieces which were randomly distributed into preweighed incubation flasks so that each came to contain 90 to 130 mg of tissue . One sample was used for LPL assay while the others (8 to 10 in each series) were incubated with gentle shaking at 37 °C for one to three hours. After this period the tissue was assayed for LPL activity and the medium for FFA content. The basal incubation medium ~c~as Krebs-Ringer phosphate solution (KRP) with 3 g~100 ml bovine albumin. AU. additions are mentioned separately below. The LPL activity was determined with radioactive rat plasma triglycBride technique described earlier 11 , colorimetric micromethod of Novak
and the FFA was measured with the
13 .
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LIPASE SYNTHESLS
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T AB LE I Relationship of FFA Release and Lipoprotein Lipase Activity of Adipose Tissue Incubated in Vitro Pieces of adipose tissue were incubated for 3 hours in the basal medium containing the additions indicated in the following concentrations : glucose 5 mg~ml, insulin 10 mU~ml, fructose 5 mg~ml, 2-deoxyglucose 5 mg~ ml and puromycin 0 . 5 mg~ml . Mean of 5 experiments . Addition
~un~g~hr
LPL
Per cent of unincubated
FFA release ~m~g~hr
None + puromycin
0 . 32 0 . 23
16
1 . 5K 1 . 71
Glucose + puromycin
0 . 62 0 . 34
27
1 . 27 1 . 50
Insulin + puromycin
1 . 18 0, 30
51
0 . 60 0 . 61
Insulin, glucose + puromycin
2 .20 0 . 57
95
0 .23 0 . 21
Fructose
1 . 65
71
0, 63
Insulin, glucose + 2-dcoxyglucosc
2 .06
89
0 .44
Re sult s On incubation in basal medium the LPL activity of adipose tissue decreased with an approximate half-life of 1 hour so that after 3 hours the activity was 10 to 20 per cent of the original value . Addition of puromycin to the medium did not modify the decay rate which indicates that the decrease of the LPL activity reflected the true degradation rate of the enzyme and that no synthesis of new enzyme occurred under these conditions (Table I), Corresponding puromycin controls were also included in the series where the LPL synthesis was induced by different additions, and also then the final LPL activity with puromycin present was similar to that of the sample incubated in basal medium . The only exception was the incubation with both glucose and
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LIPASE SYNTHESIS
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TABLE II Effect of Lipolytic Agents on in Vitro Induction of Adipose Tissue Lipoprotein Lipase Pieces of adipose tissue were incubated for 2 hours in KRP medium with 3 g~100 ml of albumin, 5 mg~ml glucose and 10 mU~ml insulin plus the additions indicated. Thereafter the tissue was assayed for LPL activity and the medium for FFA. Each series included also a control where the glucose and insulin were omitted (uninduced residual activity). The figures are mean values from 5 experiments. LPL activity um~g~hr
Per centl inhibition )
FFArelease N.m~g~hr
None (= fully induced)
2. 62
0
0 . 14
Norepinephrine 10 ng~ml
i . 51
50
"
100 ng~ml
0. 80
1 . 39
81
5 . 50
"
500 ng~ml
0. 49
100
8 . 60
2. 35
12
0. 33
1 . 31
62
1 . 45
0 . 32
100
10 . 80
Addition
Theophylline 5 x 10 -5 M -4 " 5 x 10 M "
2 x 10 -3 M
1) On calculating the inhibition percentage of the de novo synthesis the residual LPL activity present in the uninduced sample of each series was subtracted from the value of both fully induced and corresponding experimental sample . insulin which gave a slightly higher activity with puromycin (Table I) thus suggesting that this combination might in some way retard the degradation of the enzyme . Otherwise, all the deviations from the corresponding controls can thus be taken to reflect only the synthesis of new LPL protein. Puromycin did not affect the rate of FFA release, which result is in accordance with previous experience
8
and indicates that the lipases
responsible for endogenous lipolysis have a much slower turnover than the LPL . Table I shows the in vitro induction of LPL by various additions to the basal medium . It ie evident that insulin is a more powerful inducer of LPL
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than the mere availability of glucose. When present together, glucose and insulin were able to completely maintain the preincubation activity, i. e . , to induce a synthesis rate equal to that of degradation . 2-deoxyglucose added to the insulin-glucose medium caused an insignificant inhibition of LPL synthesis and a slight increase of FFA release although the glucose metabolism was inhibited by 75 per cent (measured with glucose-U- 14C, data not given) . Fructose caused a decrease of FFA release and a definite increase of LPL activity both responses being of the same magnitude âs those obtained with insulin, In all these experiments there was a significant correlation between the degree of LPL induction and the inhibition of FFA release, In the second series of experiments the effect of increased lipolysis on the LPL induction by glucose plus insulin was determined . The results in Table II demonstrate that both norepinephrine and theophylline strongly in hibited the LPL synthesis the degree of inhibition being in good correlation to the response of FFA release . The enzyme synthesis was almost completely repressed at FFA release level exceeding 5.0 }unolea. g-l . hr -1 . Control experiments were made to check that the lipolytic agents did not interact in the determination of LPL and that no leakage of the enzyme into the medium took place, a phenomenon noted to occur for some enzymes on intense . stimulation of lipolysis 16 Discussion Although it is difficult to produce such experimental conditions that the intracellular FFA concentration is varied alone without affecting, e. g. , the glucose metabolism, it is evident from the present data that induction of the adipose tissue LPL is related to FFA release much better than to y;lucose utilization . P~irticularly the very effective repression of tlic en~yi~le
130 8
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LIPASE SYNTHESIS
synthesis obtained with lipolytic agents is difficult to explain on the basis of decreased glucose metabolism since stimulated lipolytis is accompanied by 4 an increased uptake and oxidation of glucose .
Insulin is known to increase
9 but it may the protein synthesis in fat cells also in the absence of glucose well be that its inductive action at least on LPL is mediated by the antilipolytic effect . This view is supported by the finding that a similar induction is obtained with fructose, which decreases the FFA concentration by increasing re-esterification. However, alternative explanations still remain which muet be considered in future experiments . Thus, insulin and lipolytic hormones cause opposite changea, e . g. , in the cyclic 3', 5'-AI~iP content 2 and in the pentose cycle activity
4
of fat cells.
In connection of these findings it is interesting to note that epinephrine and glucagon have been reported to inhibit the in vivo induction of liver glucokinase by glucose and insulin
10, 14 .
This effect has not been
adequately explained but in view of the known lipolytic activity of these hormones - glucagon activating also the hepatic lipase 1 - it is tempting to suggest that fatty acids or acyl-CoA can function as repressor also of enzymes other than the LPL. Studies on this point are in progress . Acknowledgement - This work was supported by research grants from Sigrid Jusélius Foundation, Emil Aaltonen Foundation, Finnish Culture Foundation and Finnish State Medical Research Council. References 1.
P. D. BEWSHER and J. ASHMORE, Biochem. Biophys. Res. Commun . 24, 431 (1966) .
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A. CHERKES and R. S. GORDON, JR . , J. Lipid Res. 1, 97 (1959) .
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LIPASE SYNTHESLS
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C. H. HOLLENBERG, Am, J . Physiol, 197, 667 (1959) .
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A. KÖRNER and hl . S. RABEN, Nature 203 ; 1287 (1964) .
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L. 'v, MILLER and P. M. BEIGELMAN, Endocrinology 81, 386 (1967)
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H. NIEI~IEYER, N. PEREZ and F' . RABAJILLE, J. Biol. Chem . 241, 4055 (1966),
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E . A. NIKKILÄ and O. PYKÄLISTÖ, Biochem. Biophys, Acta 152, 421 { 1968). --`
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E. A. NIKKILÄ, P, TORSTI and O . PENTTILÄ, MetabolisYn 12, 863 (1963) . --
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M. NOVAK, J. Lipid Res. 6, 431 (1965) .
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H. C. PITOT, C . PERAINO, N, PRIES and A. L. KENNAN, Adv, Fnzyme Reg. 2, 237 (1964) .
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U. PYKÄLLSTÖ and E, A. NII{KILÄ, Scand. J . Clin . Lab, Invcst . ly_, suppl, y5, 43 (1967,
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h4, RODBELL, J, Biol. Chem, 241, 3909 (1966),
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M. R. SALAhIAN and D . S, RO$INSON, Biochem. J. 99, 640 (1966) .
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J . D . SCHNATZ and R. H. WILLIAIMS, Diabetes 12, 174 (1963),
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D. R. WING and D. S. ROBINSON, Biochem. J. 106, 667 (1968) .
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D. R. WING, M. R. SALAMAN and D, S. ROBINSON, $iochem, J . 99, 648 (1966), --