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Design and application of a special feeder for studying enzymes of the late suckling period
ANALYTICAL
BIOCHEMISTRY
51,
637640
SHORT
Design
and
Application
Enzymes
of the
(1973)
COMMUKICATIONS
of a Special Late
Suckling
Feeder
for Studying
Period1
Changes in levels of enzymes during development of rat liver or other mammalian organs tend to occur during the fifth to first days prior to birth (late fetal period), the first day after birth (early postnatal period), and the third week of postnatal life (late suckling period) (1). Enzymes of the late suckling period undergo changes in activity which coincide in time with weaning and with ingestion of solid chow. These changes may be due to hormones, to the transition from a high lipid milk diet to a high carbohydrate laboratory chow, or to both (1). To distinguish which of these factors is involved in the observed changes, it is essential to control dietary intake at this stage of development. A major problem is that suckling rats 15 days of age or older nibble at solid chow even prior to weaning. We describe a device for preventing the ingestion of solid chow by suckling pups while permitting the dams to feed ad libitum. The effectiveness of this device is illustrated with respect to changes in activity of HMG-CoA reductase [Mevalonate:NADP oxidoreductase (acylating CoA) , EC 1 I.1.341, an enzyme of the late suckling cluster. Materials and Methods. The materials and assay conditions for rat hepatic microsomal HMG-CoA reductase activity have previously been reported (24). Results. Construction of Controlled Feeding Apparatus. A plexiglass plate, 30 X 30 X 0.3 cm, to which was sealed a 9 X 9 cm plexiglass feed cup having a 7 X 7 cm side opening served as the top of the animal’s cage (Fig. 1). The aperture was 15 cm above the cage floor, which was wire mesh and allowed spilled feed to fall through. 1 Supported by grants from the American Heart Association (#71-860) and from the Cancer Research Committee of Purdue University. Journal paper number 4717 from the Purdue University Agricultural Experiment Station. The data are from the Ph.D. thesis of Donald J. McNamara (Purdue University 1972). 637 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
638
SHORT
75n’L
DIO. -.
COMMUNICATIONS
_.250”
O.D.
it Eib 3.437"
I
ITEM 0
! 2.5 0" 9
&Od ~
ITEM
@
FIG. 1. Construction of the feeder. The apparatus, constructed of 0.125-in. thick Plexiglas, consists of a flat plate which serves as the roof of the cage and from which a feeder cup is suspended.
Efiectiveness of Feeder. Nursing dams consumed chow by standing on their hind legs to reach the aperture in the feeder. Feed intake appeared normal and the dams experienced no apparent difficulty in obtaining all the chow needed. Periodic inspection of the cages and examination of the stomach contents of sacrificed pups showed that the pups did not ingest solid chow. Weight gains were within the normal values for this age animal and did not differ whether weaned or forced to continue nursing. Application. In the course of a study on the regulation of the developmental pattern of rat hepatic HMG-CoA reductase (4), we utilized this feeding apparatus to determine the effect of late weaning on the developmental pattern of the reductase (Fig. 2). Both premature and late weaning in pups allowed access to chow were followed by a significant increase in HMG-CoA reductase activity (Fig. 2). By contrast, when X-day-old pups and their dam were placed in cages equipped with the special feeder, no increase in reductase activity was noted even after 28 days. A change in diet thus appears to trigger the observed increase in reductase, and the effectiveness of the apparatus is illustrated by the complete failure of reductase levels to rise in pups denied access to solid chow. Discussion. Greengard (1) has postulated that enzymes of the late suckling cluster should consist of two subclusters: those enzymes whose
SHORT
639
COMMUNICATIONS
AGE ( DAYS 1
FIG. 2. Effectiveness of the feeder. The 500009 microsomal pellets from individual livers were assayed in duplicate for HMG-CoA reductase activity. Control animals (0-O) were weaned at 21 days of age and early weaned animals (0-O) at 16 days of age. Late weaned controls (A-A) were kept, in standard breeding cages with a normal feed dish. Pups, 15 days old, which were not to be wcancd were placed with their dam in an open wire mesh cage equipped with the special feeder (n-0). Three rats were killed for each data point. Vertical lines represent the standard error for a total of six analyses. (Reproduced from J. B&Z. Chem. 247, 5805 (1972), by permission of the copyright owner. See Ref. 4.)
rise would be blocked by preventing intake of solid chow and those whose activity should rise irrespective of dietary conditions. To distinguish to which of these subclusters a particular enzyme belongs, the dietary intake of the suckling pup must be rigidly controlled. HMG-CoA reductase appears to belong to the former subcluster. The feeding apparatus described can be utilized to prevent intake of solid chow by developing rats and thus facilitate classification of enzymes which undergo changes during the late suckling period. ACKNOWLEDGMENTS I thank Professor Victor Rodwell and Professor advice concerning the design and application of for breeding and caring for the animals.
Forrest Quackenbush this device and Lee
for helpful Sondgeroth
REFERENCES 1. GREENGARD, 0. (1970). in Biochemical p. 53, Academic Press, New York. 2. SHAPIRO, D. J., IMBLUM, R. L., AND 383. 3. SHAPIRO, D. J., AND RODWELL, V. W.
Action RODWELL,
(1971).
of
Hormones,
(G.
V.
W.
Anal.
J. Biol.
(1969). Chem.
246,
Litwack,
3210.
Biochem.
Ed.), 31,
640
SHORT COMMUNICATIONS D. J.,
4. MCNAMAFCA,
Chem.
247,
QUACKENBUSH,
F. W.,
AND
RODWELL,
V. W. (1972). J. Biol.
5805.
DONALD
J. MCNAMARA~
Department of Biochemistry Purdue University Lafayette, Indiana 47907 Received
April
Accepted
September
*Present University,
12, 182 20, 1976
address: Department of Physiological Columbus, Ohio 43210.
Chemistry,
The
Ohio
State
BIOCHEMISTRY
51,
637640
SHORT
Design
and
Application
Enzymes
of the
(1973)
COMMUKICATIONS
of a Special Late
Suckling
Feeder
for Studying
Period1
Changes in levels of enzymes during development of rat liver or other mammalian organs tend to occur during the fifth to first days prior to birth (late fetal period), the first day after birth (early postnatal period), and the third week of postnatal life (late suckling period) (1). Enzymes of the late suckling period undergo changes in activity which coincide in time with weaning and with ingestion of solid chow. These changes may be due to hormones, to the transition from a high lipid milk diet to a high carbohydrate laboratory chow, or to both (1). To distinguish which of these factors is involved in the observed changes, it is essential to control dietary intake at this stage of development. A major problem is that suckling rats 15 days of age or older nibble at solid chow even prior to weaning. We describe a device for preventing the ingestion of solid chow by suckling pups while permitting the dams to feed ad libitum. The effectiveness of this device is illustrated with respect to changes in activity of HMG-CoA reductase [Mevalonate:NADP oxidoreductase (acylating CoA) , EC 1 I.1.341, an enzyme of the late suckling cluster. Materials and Methods. The materials and assay conditions for rat hepatic microsomal HMG-CoA reductase activity have previously been reported (24). Results. Construction of Controlled Feeding Apparatus. A plexiglass plate, 30 X 30 X 0.3 cm, to which was sealed a 9 X 9 cm plexiglass feed cup having a 7 X 7 cm side opening served as the top of the animal’s cage (Fig. 1). The aperture was 15 cm above the cage floor, which was wire mesh and allowed spilled feed to fall through. 1 Supported by grants from the American Heart Association (#71-860) and from the Cancer Research Committee of Purdue University. Journal paper number 4717 from the Purdue University Agricultural Experiment Station. The data are from the Ph.D. thesis of Donald J. McNamara (Purdue University 1972). 637 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
638
SHORT
75n’L
DIO. -.
COMMUNICATIONS
_.250”
O.D.
it Eib 3.437"
I
ITEM 0
! 2.5 0" 9
&Od ~
ITEM
@
FIG. 1. Construction of the feeder. The apparatus, constructed of 0.125-in. thick Plexiglas, consists of a flat plate which serves as the roof of the cage and from which a feeder cup is suspended.
Efiectiveness of Feeder. Nursing dams consumed chow by standing on their hind legs to reach the aperture in the feeder. Feed intake appeared normal and the dams experienced no apparent difficulty in obtaining all the chow needed. Periodic inspection of the cages and examination of the stomach contents of sacrificed pups showed that the pups did not ingest solid chow. Weight gains were within the normal values for this age animal and did not differ whether weaned or forced to continue nursing. Application. In the course of a study on the regulation of the developmental pattern of rat hepatic HMG-CoA reductase (4), we utilized this feeding apparatus to determine the effect of late weaning on the developmental pattern of the reductase (Fig. 2). Both premature and late weaning in pups allowed access to chow were followed by a significant increase in HMG-CoA reductase activity (Fig. 2). By contrast, when X-day-old pups and their dam were placed in cages equipped with the special feeder, no increase in reductase activity was noted even after 28 days. A change in diet thus appears to trigger the observed increase in reductase, and the effectiveness of the apparatus is illustrated by the complete failure of reductase levels to rise in pups denied access to solid chow. Discussion. Greengard (1) has postulated that enzymes of the late suckling cluster should consist of two subclusters: those enzymes whose
SHORT
639
COMMUNICATIONS
AGE ( DAYS 1
FIG. 2. Effectiveness of the feeder. The 500009 microsomal pellets from individual livers were assayed in duplicate for HMG-CoA reductase activity. Control animals (0-O) were weaned at 21 days of age and early weaned animals (0-O) at 16 days of age. Late weaned controls (A-A) were kept, in standard breeding cages with a normal feed dish. Pups, 15 days old, which were not to be wcancd were placed with their dam in an open wire mesh cage equipped with the special feeder (n-0). Three rats were killed for each data point. Vertical lines represent the standard error for a total of six analyses. (Reproduced from J. B&Z. Chem. 247, 5805 (1972), by permission of the copyright owner. See Ref. 4.)
rise would be blocked by preventing intake of solid chow and those whose activity should rise irrespective of dietary conditions. To distinguish to which of these subclusters a particular enzyme belongs, the dietary intake of the suckling pup must be rigidly controlled. HMG-CoA reductase appears to belong to the former subcluster. The feeding apparatus described can be utilized to prevent intake of solid chow by developing rats and thus facilitate classification of enzymes which undergo changes during the late suckling period. ACKNOWLEDGMENTS I thank Professor Victor Rodwell and Professor advice concerning the design and application of for breeding and caring for the animals.
Forrest Quackenbush this device and Lee
for helpful Sondgeroth
REFERENCES 1. GREENGARD, 0. (1970). in Biochemical p. 53, Academic Press, New York. 2. SHAPIRO, D. J., IMBLUM, R. L., AND 383. 3. SHAPIRO, D. J., AND RODWELL, V. W.
Action RODWELL,
(1971).
of
Hormones,
(G.
V.
W.
Anal.
J. Biol.
(1969). Chem.
246,
Litwack,
3210.
Biochem.
Ed.), 31,
640
SHORT COMMUNICATIONS D. J.,
4. MCNAMAFCA,
Chem.
247,
QUACKENBUSH,
F. W.,
AND
RODWELL,
V. W. (1972). J. Biol.
5805.
DONALD
J. MCNAMARA~
Department of Biochemistry Purdue University Lafayette, Indiana 47907 Received
April
Accepted
September
*Present University,
12, 182 20, 1976
address: Department of Physiological Columbus, Ohio 43210.
Chemistry,
The
Ohio
State