Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver

ARCHIVES OF BIOCHEMISTRY Vol. 209, No. 2, July, pp.

Effects

AND BIOPHYSICS 6’77-681,1981

of Feeding and Lighting Stimuli on the Synthesis of Ornithine Aminotransferase and Serine Dehydratase in Rat Liver’** KAREN

Division

of Biologkal

B. EKELMAN

and Medical

Research, Received

AND Argonne

CARL National

January

PERAIN03 Laboratory,

Argonne,

Illinois

60499

5, 1981

In a previous study we showed that rats fed ad libitum and maintained on a 12-h light/ 12-h dark cycle demonstrated out-of-phase circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis. As part of an investigation of the factors regulating both the generation of these cycles and their dissimilarity, this paper compares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/lZh dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/l2-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis.

Previous studies in this laboratory have compared changes in the activities and synthesis rates of rat liver ornithine aminotransferase (L-ornithine:2-oxoacid aminotransferase, EC 2.6.1.13) and serine dehydratase (L-serine hydrolyase (deaminating), EC 4.2.1.13) in response to a variety of regulatory stimuli (l-4). These amino acid catabolizing enzymes exhibit similar responses to some regulatory stimuli: enzyme activities increase severalfold ’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. ‘Supported by the U. S. Department of Energy under Contract W-31-109-ENG-38. 3To whom requests for reprints should be addressed. 677

when rats are placed on a high-protein diet; synthesis rates for both enzymes in rats maintained on a high-protein diet oscillate with a circadian periodicity, and the phasing of these circadian enzyme oscillations is unaltered by glucocorticoid administration. Ornithine aminotransferase and serine dehydratase also exhibit dissimilar adaptive properties, however. Administration of a glucocorticoid to rats maintained on a high-protein diet decreases ornithine aminotransferase synthesis and activity, but causes serine dehydratase synthesis and activity to increase. In addition, the circadian synthesis oscillations for the two enzymes seen in rats maintained on a high protein diet are out of phase with each other (4). The existence of both similar and dissim0003~9861/81/08067’7-05$02.00/O Copyright All rights

@ 1981 by Academic Press, Inc. of reproduction in any form reserved

678

EKELMAN

AND PERAINO

ilar responses of ornithine aminotransferase and serine dehydratase to regulatory stimuli suggests that these enzymes constitute a useful model system for studying the mechanisms of enzyme regulation in rat liver. The present investigation was undertaken to characterize further the regulatory processes that are responsible for the circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis. Because circadian alterations in lighting and food intake are known to influence a variety of biochemical responses (5-lo), effort was focused on an analysis of the effects of the light/dark cycle and of food availability on the circadian synthesis oscillations for these two enzymes. MATERIALS

AND METHODS

Mute&&. Male Sprague-Dawley rats, age 21 and 42 days, were purchased from Charles River Laboratories, Wilmington, Massachusetts, and were housed two per cage under conditions of constant temperature (23°C) and either constant light or a light/dark cycle (light ‘7 AM-~ PM; dark ‘7 PM-'7 AM). Water and a standard laboratory diet (Wayne Lab Blox, Allied Mills, Chicago, Ill.) were available ud &turn. A nutritionally adequate pelleted diet containing 60% casein was purchased from Teklad Mills, Madison, Wisconsin. L-[4,5-3H@ucine, 52 Ci/mmol, was purchased from ICN Pharmaceuticals, Irvine, California. All other reagents were of the highest purity commercially obtainable, and all reagent solutions were prepared with glass-distilled water. Treatments for regulation studies. One group of rats at 23 days of age was randomized by weight (range, 121 to 182 g; average 156 g) and subjected to a 12-h light (normal room light)/l2-h dark (room lights off) cycle and restricted access (8 ~~-10 AM or 8 ~~-10 PM) to the standard laboratory diet for 11 days, followed by restricted access to a 60% casein diet for 5 days prior to sacrifice. The experiment using these rats was performed in September. Another group of rats, 49 days old (weight range, 221 to 305 g; average 251 g), was treated as described above except that some of the rats were maintained under constant light, and all rats received the 60% casein diet for 3 days prior to sacrifice. The experiments using these rats were performed in December. Additional details of treatment for each experimental group are presented in appropriate figure legends. In all cases experimental Day 0 is considered as the day on which the restricted feeding and lighting protocol was instituted.

During the sacrifice intervals (Days 14 and 16) the rats were maintained on the foregoing protocols and randomly selected rats from each treatment group were sacrificed every 4 h over the next 24 h. Forty minutes prior to sacrifice each rat was given 100 pCi of [3H]leucine in 0.1 ml by intraperitoneal injection. Rats were killed by cervical dislocation; livers were rapidly removed, chilled in ice-cold buffer (1.0 M Tris/ 0.15 M KCl/10e3 M dithiothreitol/10-3 M EDTA/10m4 pyridoxal5’-phosphate, pH 8.0), weighed, and frozen in liquid Nz. The frozen livers were individually wrapped in aluminum foil and stored in liquid Nz until analysis. Measurements of synthesis rates. Rates of ornithine aminotransferase and serine dehydratase synthesis were measured immunochemically as described previously (4) using goat antibodies prepared against thrice-crystallized preparations of each enzyme. The only procedural modification used in the present study was the inclusion of KC1 at a concentration of 0.15 M in the liver homogenizing buffer. This addition stabilized serine dehydratase during heating (4) and had no effect on the catalytic activity of either enzyme. As a consequence, purified carrier serine dehydratase was not required in the immunoprecipitation reaction (4), and correction for heatinduced loss of enzyme in the immunochemical estimation of serine dehydratase synthesis rates was not necessary. As before (4), synthesis rates were expressed as the ratio of the radioactivity @H]leucine) incorporated in 40 min in the immunoprecipitate of each enzyme to the radioactivity in the total liver protein. Changes in this ratio were considered to represent changes in the rate of synthesis of the specific enzyme relative to changes in the rate of total protein synthesis. This method of expression corrected for fluctuations in the size of the free leucine pool and for random variations in the uptake of [3H]leucine among different rats. No treatment-related change in leucine incorporation into total protein was observed in the current study. All values are expressed as means and standard errors. The numbers of animals per data point are shown in parenthesis in the figures. The vertical bars at all data points indicate one standard error. RESULTS

In rats exposed to constant light, a 2-h restricted mealtime produced reciprocal circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis (Fig. 1); ornithine aminotransferase synthesis began to increase shortly after the feeding interval and reached a maximum 12 h later, whereas serine dehydratase showed the

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AMINOTRANSFERASE,

SERINE

DEHYDRATASE

REGULATION

679

opposite response. Under these conditions the persistence of reciprocal oscillations was unaffected by the temporal location of the restricted feeding interval. When the restricted feeding interval was combined with a 12-h light/lZh dark cycle, however, the maintenance of oscillations in ornithine aminotransferase and serine dehydratase synthesis depended on the temporal location of the feeding interval within the light/dark cycle (Figs. 2 and 3). Exposure of rats to the restricted feeding interval at either the beginning of the dark phase or the beginning of the light phase produced reciprocal modifications in the synthesis rates for these

SERINE

ORNITHINE

LO

FIG. 2. Synthesis rates of ornithine aminotransferase (panel A) and serine dehydratase (panel B) measured over 24-h intervals (solid lines) in rats maintained under a 12-h light (7 AM-? PM)/lZ-h dark photoperiod and fed from 8 to 10 PM. Experimental values obtained from rats previously maintained on a 60% casein diet for 3 days (solid circles) or 5 days (open circles) prior to sacrifice are presented. The zero time reference is 9 AM; solid horizontal bars indicate periods of food availability; shaded horizontal bars indicate intervals of darkness; the data connected by dashed lines from 24 to 48 h are reiterations of zero to 24-h and 48- to 72-h experimental values, and facilitate visualization of cyclic fluctuations. The rats fed the 60% casein diet for 3 days were 49 days old and those fed this diet for 5 days were 28 days old on Day 0 of the experiment (see Materials and Methods).

DEkYDRATASE

AMINOTRANSFERASE

24 HOURS

48

FIG. 1. Synthesis rates of ornithine aminotransferase and serine dehydratase measured over a 24-h interval (solid lines) in rats maintained under constant light and fed from 8 to 10 PM (panels A and B) or from 8 to 10 AM (panels C and D). Rats (49 days old on Day 0 of the experiment-see Materials and Methods) were fed a 60% casein diet for 3 days prior to sacrifice. The zero time reference is 9 AM; solid horizontal bars indicate intervals of food availability; the data connected by dashed lines are reiterations of the zero to 24-h experimental values, and facilitate visualization of cyclic fluctuations.

enzymes. When the feeding stimulus was presented at the beginning of the dark phase (Fig. 2) serine dehydratase synthesis oscillated (Fig. 2B) as it had in rats maintained on constant light (Figs. 1B and D), but the oscillations in ornithine aminotransferase synthesis were eliminated (Fig. 2A) and the rate of ornithine aminotransferase synthesis was sustained at high levels approximating the peak values attained when the synthesis of this enzyme was oscillating (Figs. 1A and C; Fig. 3A). In contrast, presentation of the feeding stimulus at the beginning of the light phase (Fig. 3) was accompanied by oscillations in ornithine aminotransferase synthesis (Fig. 3A) similar to those in rats maintained on constant light (Figs. 1A

680

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AND PERAINO

circles in Figs. 2 and 3) food consumption and body weight gain for rats fed between 9 AM and 10 AM were 17.1 +: 0.3 g and 2.5 + 0.1 g, respectively (number of rats, 55), whereas those fed between 8 PM and 10 PM consumed 18.5 f 0.3 g and gained 2.7 + 0.1 g per day (number of rats, 35). It is clear, therefore, that food consumption and body weight gain were essentially unaffected by any of the feeding and lighting changes to which the rats were subjected. DISCUSSION

FIG. 3. Synthesis rates of ornithine aminotransferase (panel A) and serine dehydratase (panel B) measured over 24-h intervals (solid lines) in rats maintained under a 12-h light (7 AM-7 PM)/l&h photoperiod and fed from 8 to 10 AM. The remaining descriptive information is as indicated in the legend for Fig. 2.

and C) whereas the oscillations in serine dehydratase synthesis were eliminated (Fig. 3B). Moreover, the nonoscillating levels of serine dehydratase synthesis remained low, approximating the minimum values observed when the synthesis of this enzyme was oscillating (Figs. 1B and D; Fig. 2B). Note that the response patterns of the two groups denoted by the solid and open circles in Figs. 2 and 3 were similar despite the differences between these two groups with respect to the duration of exposure to the 60% casein diet, the ages and body weights of the rats, and the time of year at which the experiment was performed. Average daily food consumption and body weight gain were monitored, during the 11-day acclimation interval preceding the feeding of the 60% casein diet, both in rats on constant light and in those exposed to the light/dark cycle. Under constant light (Fig. 1) rats fed from 8 AM to 10 AM ate 16.5 + 0.2 (SE) g and gained 2.0 f 0.2 g per day (number of rats, 56). The rats fed from 8 PM to 10 PM ate 16.8 f 0.2 g and gained 1.9 + 0.1 g per day (number of rats, 55). Under the light/dark cycle (data from the rats denoted by the open

The results of this investigation indicate that both food ingestion and the light/ dark cycle are actively involved in the control of rat liver ornithine aminotransferase and serine dehydratase synthesis. Thus, the reciprocal cyclic responses of ornithine aminotransferase and serine dehydratase synthesis rates to meal feeding in rats under constant light (Fig. 1) indicate that food ingestion per se produces a transient stimulation of ornithine aminotransferase synthesis and a simultaneous transient repression of serine dehydratase synthesis. The regulatory influence of fluctuations in illumination on the synthesis rates for both enzymes is clearly demonstrated by the observed modulations of the effects of food ingestion on enzyme synthesis, when the feeding interval was superimposed on a light/ dark cycle (Figs. 2A and 3B). With respect to the mechanism(s) of the feeding effects, it is not yet known whether the adaptive responses noted above are mediated directly by dietary components or indirectly via the actions of endogenous regulators stimulated by the feeding event. The effects of the light/dark cycle on enzyme synthesis are evidently mediated by endogenous regulatory factors arising as a consequence of the excitation of appropriate nuclei within the central nervous system by the onset of illumination (11, 12). The identification of the meal- and light-responsive factors and the determination of their sites of action will be undertaken in subsequent investigations. In the absence of such information, however, it is still possible to gain under-

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standing of the manner in which these factors interact in the regulation of ornithine aminotransferase and serine dehydratase synthesis by examining the adaptation patterns shown in Figs. 2 and 3. The simplest scheme that can explain these observations involves the interactions of three labile regulatory factors; a meal-responsive ornithine aminotransferase inducer, a meal-responsive serine dehydratase repressor, and an inducer for both enzymes that is stimulated by the dark/light transition. (The required number of separate factors would be reduced to two if evidence for the identity of the meal-responsive regulators for the two enzymes was obtained.) According to the proposed scheme, when the feeding interval is located at the beginning of the dark phase of the light/dark cycle the meal- and light-responsive inductive stimuli for ornithine aminotransferase are out of phase with each other thereby providing a relatively constant positive signal that results in a continuous high rate of synthesis for this enzyme. Under these conditions the meal-responsive repressive signal and light-responsive inductive stimulus for serine dehydratase are also out of phase with each other, thereby providing alternating inductive and repressive signals that lead to the oscillations of serine dehydratase synthesis seen in Fig. 2B. When the feeding interval is located at the beginning of the light phase, the mealand light-responsive inductive stimuli for ornithine aminotransferase are in phase with each other. This overlap is followed by an interval during which no inducer is present, and this alternating sequence results in oscillations of ornithine aminotransferase synthesis (Fig. 3A). The low nonoscillating level of serine dehydratase synthesis observed under these conditions (i.e., when the meal-responsive serine dehydratase repressor is in phase with the light-responsive inductive stimulus for this enzyme) suggests that the repressive

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DEHYDRATASE

681

REGULATION

effect of meal feeding dominates the inductive effect of the dark-light transition. The existence of such a regulatory hierarchy requires either differential affinities of the inducer and repressor for a common regulatory site, or different sites of action for the inducer and repressor, with the repressor acting nearer the terminal stages of protein synthesis. Support for the latter possibility is provided by our previous observation that the glucocorticoid-mediated repression of ornithine aminotransferase synthesis occurs at a post-transcriptional stage in the synthesis of the enzyme (13). ACKNOWLEDGMENTS Excellent technical Aldona Prapuolenis.

assistance

was provided

by Ms.

REFERENCES

1. PERAINO,

C. (1967)

3. RAHMAN,

BioL Chem. 242.3360-3367. B&him. Biqphys. Acta 165.

J.

2. PERAINO, C. (1968) 108-112.

Y. E., ANDPERAINO,

C. (19’73)

Exp. Ger-

onto1. 8. 93-100. 4. MORRIS,

J. E., AND PERAINO,

C. (19’76)

J.

Bid

Chem. 251.25’712578. 5. HALBERG,

F., VISSCHER, M., AND BITTNER, J. (1953) Amer. J. PhysioL 174,1&l-122. 6. HALBERG, F., GALICHICH, J. H., UNGAR, F., AND FRENCH, L. A. (1965) Proc. Sot. Exp. BioL Med. 118.414-419. 7. HAUS, E., AND HALBERG, F. (1966) Ezperientia 22,113-114.

8. PIER, (1966)

V.,

GEBERT,

R. A.,

AND PITOT,

H.

C.

Advan. Enzyme ReguL 4.247-265. 9. EHRET, C. F., AND PO’ITER, V. R., (1974) J. Chronobiol.

2.321-326.

10. PHILIPPENS, K. M. H., VON MAYERSBACH, H., AND SCHEVING, L. E. (1977) J. N&r. 107, 176-193. 11. GROOS. G., AND MASON, R. (1978) Neurosci. Lett.

8.5944. 12. RUSAK.

B., AND ZUCKER,

I. (1979)

PhgsioL Rev.

59,449-526. 13. PERAINO, (1976)

C., MORRIS, J. E., AND SHENOY, Life Sci. 19.1435-1438.

S. T.