Hormonal control of specific gene expression in the rat liver during the suckling-weaning transition

HORMONAL CONTROL OF SPECIFIC GENE EXPRESSION IN THE RAT LIVER DURING THE SUCKLING-WEANING TRANSITION DOMINIQUE P E R D E R E A U , MICHAEL NARKEWICZ, CHRISTINE COUPE, PASCAL FERRE and JEAN G I R A R D Centre de Recherche sur la Nutrition, CNRS, 92190 Meudon-Bellevue, France

INTRODUCTION

In the rat, the suckling-weaning transition is accompanied by marked changes of nutrition. During the suckling period, the pups are fed with milk which is a high-fat low-carbohydrate diet (1). At weaning, milk is progressively replaced by the rat chow which is a high-carbohydrate low-fat diet (1). This is attended by considerable hormonal modifications: an increase in plasma insulin and a decrease in plasma glucagon concentrations (2), as well as by marked changes in the activity of rate-limiting enzymes of metabolic pathways in liver: decrease in phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (3) and of gluconeogenesis (4-6), increase in ATP-citrate lyase (EC 4.1.3.8) (7-10), acetyl-CoA carboxylase (EC 6.4.1.2) (11-13), fatty acid synthetase (EC 2.3.1.85) (14-16) and lipogenesis (7, 8, 13, 14, 16, 17) and appearance of glucokinase (EC 2.7.1.1) (18-19). However, there are few data available concerning the precise time-course of changes in these enzymes, their dietary and hormonal regulations and the molecular mechanisms involved (transcriptional or post-transcriptional regulation). The recent availability of specific cDNA probes for phosphoenolpyruvate carboxykinase (20), acetyl-CoA carboxylase (21), fatty acid synthase (22) and glucokinase (23) has prompted us to study the role of pancreatic hormones and of nutrition in the changes of the expression of these genes at weaning in the rat.

MATERIALS

AND METHODS

Animals. Female Wistar rats bred in our laboratory were used. They were

housed in plastic cages at a constant temperature (24°C). For experiments on 15- to 30-day-old rats, a light-dark cycle was used (light on from 3 p.m. to 3 a.m.) in order to sample tissues during the absorptive state of animals at the time of experiments (between 9 a.m. and 10 a.m.) (24). The studies were performed on 15- and 21-day-old suckling rats, and 22-, 23-, 25- and 91

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30-day-old weaned rats. The litters were standardized at birth to ten animals. The rats were weaned at 21 days to a semi-synthetic high-carbohydrate diet [72% carbohydrate, 1% fat, 27% protein (wt/wt)] or a high-fat diet [<1% carbohydrate, 72% fat, 28% protein (wt/wt)]. Since suckling rats begin to nibble the food of the mother at 15 days of age (1), the mothers were fed a high-fat diet from the 14th day of lactation until weaning to avoid any consumption of carbohydrates by the pups between 15 and 21 days of age. Animals were killed by decapitation between 9 a.m. and 10 a.m., i.e., in the absorptive state. Blood was collected and plasma was kept frozen at -20°C for plasma insulin and glucagon determinations. Liver was immediately removed, frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction and enzyme activity measurements.

Force-feeding studies. For force-feeding experiments, light was switched on from 7 a.m. to 7 p.m. The 21-day-old pups were removed from their mother at 7 a.m. and were kept unfed for 3 hr before the experiment begins. Force-feeding was done with the carbohydrate portion of the high-carbohydrate diet (1 g of starch and 300 mg of sucrose dissolved in 2 ml of water). Control animals received 2 ml of water. Animals were killed by decapitation before or 30 min, 1, 2, 4, and 6 hr after force-feeding. At each time, blood samples were collected and treated as described below for determination of plasma insulin, glucagon and glucose concentrations. At 0, 30 min, 1, 2, 4 and 6 hr, the liver was removed and treated as indicated below. Analytical Techniques Blood glucose. Blood samples were treated as described previously (25) and blood glucose was measured by the glucose oxidase method (kit from Boehringer Mannheim, Meylan, France). Plasma insulin and glucagon. Blood samples were centrifuged at 4°C in the presence of heparin and 20 ttl of Iniprol (peptidase inhibitor, 200,000 Units/ml, Laboratoire Choay, Paris, France). Plasma insulin and glucagon were determined by radioimmunoassay as described previously (25). Enzyme activities. The activities of phosphoenolpyruvate carboxykinase, fatty acid synthase and acetyl-CoA carboxylase were determined as described previously (26). The activity of glucokinase was determined according to Iynedjian et al. (23). Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Lab., Miinchen, West Germany), with bovine serum albumin as standard.

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Liver R N A extraction. Frozen samples of liver were pulverized in liquid nitrogen and R N A were extracted using the hot phenol procedure (26, 27). R N A concentration was determined by spectrophotometry at 260 nm and their integrity was systematically checked by electrophoresis in a 0.8% agarose submarine minigel stained with ethidium bromide and visualized under ultraviolet fluorescence. Northern and dot-blot analysis of total RNA. Electrophoresis was done in 1% agarose gels after denaturation of R N A with formaldehyde (28). RNA was transferred to a nylon membrane (Hybond-N filters, Amersham, France) by capillary transfer (29). After transfer, the RNA was fixed to the membrane by ultraviolet irradiation. R N A molecular size was estimated with ethidium bromide staining of molecular weight standards (RNA ladder) of 9.5, 7.5, 4.4, 2.4, 1.4, and 0.24 Kb (Bethesda Research Laboratories, Gaithersburg, MD, USA). The filters were kept at 4°C until hybridization. Dot-blots of RNA were made with the same type of membrane. cDNA probes were labeled with [et-3zp]dCTP using Multiprime DNA labeling system (Amersham, Bucks, UK) to a specific activity of 1-2 x 109 dpm//zg insert (30). Procedures for RNA transfer, dot-blot and hybridization were according to the instructions of the membrane manufacturer. For quantification of hybridizable RNA, autoradiograms of dot-blots or of Northern blots were scanned with a densitometer (Hoeffer Scientific Instruments, San Francisco, CA, USA). The correlation coefficient (R) between the Northern and the dot-blot procedures was > 0.90 for all enzymes, indicating that the changes in the relative concentrations of these specific mRNA were appreciated to the same extent using one or the other procedure. cDNA probes. The pPCK10 cDNA for cytosolic phosphoenolpyruvate carboxykinase mRNA is 2,600 bp in length, inserted into the PstI site of pBR 322 (20), and was provided by Dr. R. W. Hanson (Cleveland, Ohio, USA). The P181-6 cDNA for acetyl-CoA carboxylase mRNA is 509 bp in length, inserted into EcoRI site of pGEM3 (21), and was obtained from Dr. K. H. Kim (West Lafayette, Indiana, USA). The pFAS 18 cDNA for fatty acid synthase mRNA is 660 bp in length, inserted into PstI site of pBR 322 (22), and was obtained from Dr. A. G. Goodridge (Iowa City, Iowa, USA). The pUC-GK1 for glucokinase mRNA is 1,800 bp in length, inserted into the EcoRI site of pUC13 (23), and was provided by Dr. P. B. Iynedjian (Gen6ve, Suisse). Materials. Chemicals of the highest purity grade were purchased from Sigma (St. Louis, MO, USA), Farmitalia (Carlo Erba, Milano, Italia)

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or Prolabo (Paris, France). Nylon Hybond-N filters, Multiprime DNA labeling system, [ot-32p]dCTP (specific activity 3,000 Ci/mmol), and Hyperfilms MP were supplied by Amersham International (Amersham, Bucks, UK). Chemicals for enzyme assays were from Boehringer Mannheim (Meylan, France); NaH[14C]O3 (54.3 mCi/mmol) was purchased from Centre de l'6nergie atomique (Saclay, France). Statistical analysis. Results are expressed as means + SE. Statistical analysis was performed by Student's t test for unpaired data.

RESULTS Time-Course of Changes in m R N A Concentrations and Enzyme Activities after Weaning to a High-Carbohydrate Diet In order to determine the kinetics of the increase of fatty acid synthase, acetyl-CoA carboxylase and glucokinase mRNA, and the disappearance of phosphoenolpyruvate carboxykinase mRNA that occur at weaning to the high-carbohydrate diet, suckling rats were artificially weaned at 21 days of age to a high-carbohydrate diet. Since suckling rats begin to nibble the mother's food from 15 days of age (1), the mothers were fed a high-fat diet, and we verified that nibbling high-fat diet by the pups did not modify the parameters studied. The changes of fatty acid synthase, acetyl-CoA carboxylase and phosphoenolpyruvate carboxykinase mRNA concentrations between 15 and 21 days were not significant (results not shown). The glucokinase mRNA concentration slightly increases between 15 and 21 days in suckling rats. The changes in plasma insulin concentration, fatty acid synthase, acetyl-CoA carboxylase, glucokinase and phosphoenolpyruvate carboxykinase mRNA concentrations and enzyme activities during an artificial weaning on a high-carbohydrate diet are presented in Figures 1, 2 and 3. Weaning to a high-carbohydrate diet results in a rise of plasma insulin concentration and a fall of plasma glucagon (Fig. 1). The plasma insulin concentration, low just before weaning, is increased 2-fold 24 hr later (p < 0.05) and then remains high until 25 days. Fatty acid synthase, acetyl-CoA carboxylase and glucokinase mRNA concentrations on the one hand, and phosphoenolpyruvate carboxykinase mRNA concentration on the other hand, show totally opposite time-course (Figs. 2 and 3). Fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations, expressed in % of the maximum value, are barely detectable before weaning, and reach respectively 55% and 39% of the maximal value in the

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GENE EXPRESSION DURING DEVELOPMENT PLASMAINSULINANDGLUCAGONeONCEICrRATIONS DURINGTHE SUCKLING°WEaI~III,~ITRANSITION

INSULIN (l~U/ml)

GLUCAGON (pglml)

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21

22

23

25 clays

I Weani~ on a hiQh-carboh)K:lratedietI

FIG. 1. Plasma insulin and glucagon concentrations after weaning to a high-carbohydrate diet. Values are the mean + SE of 4-7 determinations.

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FIG. 2. Fatty acid synthase and acetyl-CoA carboxylase m R N A and activities after weaning to a high-carbohydrate diet. The results are presented in % of maximal value observed at 25 days. Values are the mean + SE of 4-7 determinations.

first 24 hr following weaning to a high-carbohydrate diet (Fig. 2). Values are respectively 66% and 73% after 48 hr and are maximal 96 hr after weaning to a high-carbohydrate diet (Fig. 2). Glucokinase mRNA concentrations, expressed in % of the maximum value, are barely detectable before weaning, and reach a maximal value in the first 24 hr following weaning to a high-carbohydrate diet (Fig. 3). Values decrease to respectively 90% and 70%, 48 hr and 96 hr after weaning to a high-carbohydrate diet (Fig. 3).

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:oL

mRNA

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FIG. 3. Phosphoenolpyruvate carboxykinase and glucokinase mRNA and activities after weaning to a high-carbohydratediet. The results are presented in % of maximalvalue observed at 21 days for phosphoenolpyruvate carboxykinase and 22 days for glucokinase. Values are the mean + SE of 4-7 determinations except for glucokinase activity which is the mean of 2 determinations. In contrast, phosphoenolpyruvate carboxykinase m R N A concentration falls to its lowest value (13 ___ 2%) in the first 24 hr. The value then remains very low onwards. Enzyme activities follow a pattern similar to that of m R N A but with a lag period of about 24 hr for fatty acid synthase and acetyl-CoA carboxylase, suggesting that transcriptional regulation plays an important role in setting activity levels (Figs. 2 and 3). Influence o f the Diet on m R N A Concentrations and E n z y m e Activities in Weaned Rats In order to investigate the influence of the diet on the changes of m R N A concentrations and enzyme activities occurring at weaning in the rat, these parameters were measured in 15-day-old suckling rats and 30-day-old rats, weaned at 21 days to a high-carbohydrate or a high-fat diet. Weaning to a high-carbohydrate diet. Fatty acid synthase, acetyl-CoA carboxylase and glucokinase m R N A are barely detectable in the liver of 15-day-old suckling rats, and are markedly increased after HC-weaning, whereas phosphoenolpyruvate carboxykinase m R N A presents a totally reciprocal variation (Figs. 4 and 5). Fatty acid synthase, acetyl-CoA carboxylase and glucokinase relative m R N A concentrations are respectively 16-, 6- and 6-fold higher in HC-weaned rats than in suckling rats (Figs. 4 and 5). Conversely, phosphoenolpyruvate carboxykinase m R N A concentration is 5-fold lower after weaning to the H C diet (Fig. 5).

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GENE EXPRESSION DURING DEVELOPMENT

FATTY ACIDSYNTHETASE I mRNA Activity

IACETYL-CCARBOXYLASE oA mRNA

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FIG. 4. Fatty acid synthase and acetyl-CoA carboxylase mRNA and activities after weaning to a high-carbohydrate or a high-fat diet. The suckling rats (S) are 15 days old. The weaned rats are 30 days old and have been weaned to high-carbohydrate (HC) or high-fat (HF) diet at 21 days. Values are the mean + SE of 4-7 determinations.

EFFECT OFWEANN ION GAHG t H-CARBOHYOR RATHIEGH-FDI ATEON T PHOSPHOENOLPYRUVATE CANBOXYKN IAND ASG ELUCOKN I mRNA ASEANDENZYM ACTI E VITIES P] HOSPHOENOLPYN CUAVRABTOEXYKN IIASE IGLUCOKIN]ASE mNNA mRNA Activity 10 30

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FIG. 5. Phosphoenolpyruvate carboxykinase and glucokinase mRNA and activities after weaning to a high-carbohydrate or a high-fat diet. The suckling rats (S) are 15 days old. The weaned rats are 30 days old and have been weaned to high-carbohydrate (HC) or , high-fat (HF) diet at 21 days. Values are the mean + SE of 7 determinations.

Weaning to a high-fat diet. Weaning to a high-fat diet prevents the increase of fatty acid synthase, acetyl-CoA carboxylase and the decrease of phosphoenolpyruvate carboxykinase mRNA concentrations and enzyme activities observed when suckling rats are weaned to a highcarbohydrate diet (Fig. 5). In contrast, weaning to a high-fat diet reduces by only 50% the increase in glucokinase mRNA and activity (Fig. 5). Feeding Carbohydrate to 21-Day-Old Suckling Rats To further assess the role of carbohydrates in the large changes of fatty acid synthase, acetyl-CoA carboxylase, glucokinase and phosphoenolpyruvate carboxykinase m R N A concentrations occurring at weaning to a HC diet, 21-day-old suckling rats were force-fed with a mixture of sucrose and starch.

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150

300

100

200

50

100

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FIG. 6. Plasma insulin and glucagon concentrations after carbohydrate force-feeding to 21-day-old suckling rats. Values are the mean + SE of 4-5 determinations.

Blood glucose, plasma insulin and glucagon concentrations are shown in Figure 6. Both glucose and plasma insulin concentrations rapidly increase from 98 + 4 mg/dl and 29 + p.U/ml in control rats (n -- 7) to reach maximal values 1 hr after carbohydrate ingestion, respectively 219 + 15 mg/dl and 109 + 30/zU/ml (n = 5). These values did not vary significantly during the 6 hr of the experiment. Plasma glucagon decreases after 3 hr and onwards (Fig. 6). The changes of fatty acid synthase, acetyl-CoA carboxylase, glucokinase and phosphoenolpyruvate carboxykinase mRNA concentrations were quantitated by scanning the dot-blots; the results, expressed in arbitrary units, are shown in Figures 7 and 8. Fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations are very low prior to high-carbohydrate force-feeding and do not increase in the 6 hr following carbohydrate force-feeding (Fig. 7). Glucokinase mRNA concentration is very low prior to carbohydrate force-feeding, rapidly increases after 2 hr and continues to rise until 6 hr (Fig. 8). Phosphoenolpyruvate carboxykinase mRNA concentration falls to 10% of the initial value in the first 2 hr following carbohydrate force-feeding, and then remains very low until 6 hr (Fig. 8). The activities of fatty acid synthase, acetyl-CoA carboxylase and glucokinase do not change during the 6 hr following carbohydrate force-feeding, whereas the activity of phosphoenolpyruvate carboxykinase decreases by 50% (results not shown). Thus, the changes in mRNA concentrations precede the changes in enzyme activities.

G E N E EXPRESSION D U R I N G DEVELOPMENT

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FIG. 8. Phosphoenolpyruvate carboxykinase and glucokinase mRNA after carbohydrate force-feeding to 21-day-old suckling rats. Values are the mean + SE of 4-5 determinations.

DISCUSSION

The aim of the present study was to investigate what are the hormonal and nutritional factors and the molecular mechanisms which might be responsible for the changes of gluconeogenesis, lipogenesis and glucokinase activity in rat liver during the suckling-weaning transition.

Lipogenic Enzyme mRNA and Activity We have confirmed that the activities of two of the major lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthetase, are low during

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the suckling period and increase at weaning to a high-carbohydrate diet (7-16). In the adult rat, short-term regulation of acetyl-CoA carboxylase is mediated by allosteric mechanisms (changes in cellular citrate concentration) and/or covalent modifications (phosphorylation/dephosphorylation) (31), whereas long-term regulation involves changes in the amount of enzyme controlled by the rates of protein synthesis and degradation (32). In the present study, we have measured the activity of acetyl-CoA carboxylase in the presence of citrate, so that we have determined its total activity. Previous studies have shown that total activity of acetyl-CoA carboxylase is directly related to the amount of enzyme (32). For fatty acid synthase, it has also been shown that the enzyme activity parallels closely the amount of enzyme (33). Thus, the increased lipogenic enzyme activities at weaning are probably linked to an increased amount of enzymes. The increases in fatty acid synthase and acetyl-CoA carboxylase activities are preceded by similar changes in the relative concentration of specific mRNA (Fig. 2), suggesting a major control of enzyme concentration at a pretranslational level. It has been suggested previously, on the basis of experiments using puromycin and actinomycin D, that protein and mRNA synthesis were a prerequisite for the increased activities of lipogenic enzymes at weaning in the mouse liver (14). More recent studies using cDNA probes have shown that the changes in fatty acid synthase and acetyl-CoA carboxylase activity in the adult rat liver were paralleled by a change in the mRNA concentration in various physiological and pathological conditions (34-36), and that the rate of lipogenic enzyme synthesis in avian liver was almost entirely dependent upon the concentration of mRNA (36). Moreover, measurement of the transcriptional rate of the fatty acid synthetase gene suggested that the dietary regulation is primarily transcriptional (36, 37). Several lines of evidence suggest that the dietary changes (rather than the developmental stage) play a major role in this phenomenon. Indeed, weaning to a high-fat diet prevents the increase in fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations (Fig. 4) and fatty acid synthase, acetyl-CoA carboxylase (11, and Fig. 4) and ATP-citrate lyase (38) activities. It has been reported that induction of diabetes with streptozotocin in 25-day-old rats suppresses the post-weaning increase in liver ATP-citrate lyase activity, and that insulin replacement therapy allows a normal increase in liver ATP-citrate lyase activity (39). Recent studies performed in adult mice have shown that the low concentration of fatty acid synthase mRNA in liver of diabetic animals was restored 6 hr after insulin injection (35, 36), suggesting a rapid action of insulin. This suggests that a relationship could exist between plasma insulin concentration and the rise of fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations.

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The sequence of events that occur after carbohydrate ingestion in suckling rats (Figs. 2 and 7) (1) increases in blood glucose and plasma insulin concentrations after 1 hr, (2) no increase in fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations during the following 6 hr, (3) 5-fold increase in fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations after 24 hr, is surprising. Indeed, fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations are increased by 10-20-fold in white adipose tissue of the same rats 6 hr after carbohydrate force-feeding (26). This could be due to several factors: (1) fatty acid synthase and acetyl-CoA carboxylase mRNA concentrations are 4-5-fold higher in white adipose tissue than in liver, and the method used for quantification of mRNA is perhaps not sufficiently sensitive to detect the early changes occurring in the liver, (2) the steps controlling the concentrations of lipogenic enzyme mRNA (transcriptional and/or post-transcriptional) are different in the liver and in adipose tissue, (3) the synthesis of one or several factors is required to stimulate lipogenic enzyme gene transcription in the liver but not in adipose tissue of suckling rats. Thyroid hormones also stimulate the expression of lipogenic enzyme mRNA in adult rat liver (40). In the present study, it is unlikely that thyroid hormones play a primary role in the changes of fatty acid synthase and acetyl-CoA carboxylase mRNA concentration, since plasma T 4 and T 3 concentrations increase rapidly to peak levels at 15-20 days after birth (41), and do not change between 21 and 30 days in rats weaned on a high-carbohydrate or high-fat diet (26). However, as the plasma concentration of thyroid hormones during weaning is similar to that of adult rat (26, 41), these hormones could play a permissive role in the induction of lipogenic enzymes (13).

Phosphoenolpyruvate Carboxykinase mRNA and Activity During the suckling-weaning transition, phosphoenolpyruvate carboxykinase activity and mRNA concentration in liver (42, Fig. 3) follow a reciprocal pattern when compared to lipogenic enzyme and glucokinase activities and mRNAs (Figs. 2 and 3). This suggests that the changes in enzyme mRNA are part of a regulatory system rather than a mere developmental event linked to cell growth or multiplication. It is also interesting to note that in the carbohydrate feeding experiments, phosphoenolpyruvate carboxykinase mRNA concentrations were rapidly and markedly decreased (Fig. 8) and that phosphoenolpyruvate carboxykinase activity is decreased by 50% 6 hr after carbohydrate feeding. These findings are not unexpected if phosphoenolpyruvate carboxykinase gene transcription is blocked after carbohydrate feeding since the half-life of phosphoenolpyruvate carboxykinase mRNA is 30 min and that of the

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enzyme is 6 hr (43, 44). This suggests that the factors responsible for the disappearance of liver phosphoenolpyruvate carboxykinase after weaning act by turning off the transcription of the gene. The nature of these factors appears to be partly related to the dietary shift from milk (a high-fat diet) to a high-carbohydrate diet. Indeed, weaning to a high-fat diet abolishes the post-weaning decrease in phosphoenolpyruvate carboxykinase mRNA concentration and activity (38, Fig. 5). In the liver of adult rats, starvation and diabetes increase the activity and synthesis of phosphoenolpyruvate carboxykinase, whereas refeeding carbohydrate to starved rats and insulin treatment of diabetic rats have the opposite effects (20, 44-46). Insulin and glucagon (via cAMP) regulate liver phosphoenolpyruvate carboxykinase by changes in the transcription rates (47-49). Recent experiments in primary culture of hepatocytes from 10-dayold rats have shown that insulin decreases in a dose-dependent fashion the concentration of phosphoenolpyruvate carboxykinase mRNA (unpublished data). This suggests that insulin is a prime negative regulator of phosphoenolpyruvate carboxykinase gene expression during the suckling-weaning transition, as in hepatoma cells (49).

Glucokinase m R N A and Activity We have confirmed that the activity of glucokinase is low during the suckling period and increases at weaning to a high-carbohydrate diet (18, 19). These changes are preceded by alterations in glucokinase mRNA (22, Figs. 3 and 5). It is also interesting to note that in the carbohydrate feeding experiments, glucokinase mRNA concentration was rapidly and markedly increased (Fig. 8). This suggests that the factors responsible for the appearance of liver glucokinase after weaning act by turning on the transcription of the gene. The nature of these factors appears to be partly related to the dietary shift from milk (a high-fat diet) to a high-carbohydrate diet. Indeed, weaning to a high-fat diet reduces the post-weaning increase in glucokinase mRNA concentration and activity (50, Fig. 5). Premature induction of glucokinase activity to 30% of adult levels can be elicited by oral glucose administration to 13-day-old rats, and it is prevented by simultaneous injection of mannoheptulose, which inhibits insulin secretion (51). Moreover, newborn rats fed a high carbohydrate formula by intragastric cannulas from the second day of life have 30% of adult glucokinase activity by day 4 and 71% of adult glucokinase activity by day 10 (52). Glucokinase activity can be induced within 4 hr by glucose (10--30 mM) and insulin (10-9-10-6 M) in incubated hepatocytes (53) or in cultured hepatocytes from 13-14-day-old rats (54). Recent experiments in primary culture of hepatocytes from 10-day-old rats have shown

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that the initial expression of mRNA of glucokinase is dependent on insulin but not on glucose concentration (55). This suggests that insulin is a prime positive regulator of glucokinase gene expression during the suckling-weaning transition, as in the adult rat. Indeed, in the adult rat, liver glucokinase mRNA concentration is low during fasting and diabetes (23, 56) and increases 50-fold within 6-8 hr of an oral glucose load to fasted rats or insulin administration to diabetic rats (23, 56). The mRNA levels return to basal values by 16-18 hr of refeeding or insulin administration, whereas the enzyme activity increases regularly until 24 hr (23, 56). The rate of transcription of the glucokinase gene is increased about 20-fold within 1-2 hr of insulin administration to diabetic rats (56, 57), and returns to the pre-stimulation level after 8 hr (56). The induction of glucokinase results primarily from a transient stimulation of transcriptional activity of the gene, leading to a short-term accumulation of glucokinase mRNA; the sustained increase in enzyme activity being due to the long half-life (30 hr) of the enzyme (56).

CONCLUSIONS The suckling-weaning transition in the rat represents a period of dramatic changes in the lipogenic, glycolytic and gluconeogenic capacities of liver. The appearance of glucokinase and lipogenic enzymes and the disappearance of phosphoenolpyruvate carboxykinase in liver after weaning to a high-carbohydrate diet could explain the development of a normal insulin sensitivity in this tissue (58). It is clear from the present study that this involves a pretranslational regulation of the synthesis of key regulatory enzymes which could be brought about by the pancreatic hormones in response to modifications of the nutritional environment.

SUMMARY In the rat, the suckling-weaning transition is accompanied by marked changes in nutrition. During the suckling period, the pups are fed with milk which is a high-fat low-carbohydrate diet. At weaning, milk is progressively replaced by the rat chow which is a high-carbohydrate low-fat diet. This is accompanied by considerable hormonal modifications: an increase in plasma insulin and a decrease in plasma glucagon concentrations, as well as by marked changes in metabolic pathways in liver: decrease in hepatic gluconeogenesis, increase in lipogenesis, and appearance of liver glucokinase. Most of the data concerning these changes are related to maximal activity of enzymes. The recent availability of specific cDNA probes for phosphoenolpyruvate carboxykinase, acetyl-CoA carboxylase, fatty

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acid synthase and glucokinase has allowed study of the role of pancreatic hormones and of nutrition in the changes of the expression of these genes at weaning in the rat. Fatty acid synthase and acetyl-CoA carboxylase m R N A in liver. The concentration of mRNA as well as the activity of acetyl-CoA carboxylase and fatty acid synthase are both very low in the liver of suckling rats. At weaning to a high-carbohydrate diet, the rapid increment in acetyl-CoA carboxylase and fatty acid synthase mRNA concentrations (10-20-fold) is followed by parallel changes in enzyme activities. In contrast, weaning on a high-fat diet prevents the increase in m R N A and activity of acetyl-CoA carboxylase and fatty acid synthase. Force-feeding suckling rats with carbohydrate is not associated with an increase in liver acetyl-CoA carboxylase and fatty acid synthase mRNA concentrations within 6 hr, despite marked increase in blood glucose and plasma insulin. This suggests that the accumulation of lipogenic enzyme mRNA in liver, 24--48 hr after carbohydrate feeding, could involve the synthesis of a factor needed for activation of gene transcription by insulin and glucose. Phosphoenolpyruvate carboxykinase m R N A in liver. The concentration of m R N A as well as the activity of phosphoenolpyruvate carboxykinase are elevated in the liver of suckling rat until the onset of weaning, 21 days after delivery. After weaning to a high-carbohydrate diet, both mRNA and activity of phosphoenolpyruvate carboxykinase rapidly decrease to a very low level. In contrast, weaning on a high-fat diet, which maintains high plasma glucagon and low plasma insulin levels, does not decrease mRNA and activity of phosphoenolpyruvate carboxykinase. Force-feeding suckling rats with carbohydrate results in increased plasma glucose and insulin concentrations and a 90% decrease in phosphoenolpyruvate carboxykinase mRNA in less than 1 hr. This suggests that hyperinsulinemia is the primary factor responsible for inhibition of liver phosphoenolpyruvate carboxykinase gene transcription. Glucokinase m R N A in liver. The concentration of mRNA as well as the activity of glucokinase are not detectable before 15 days after birth in the liver of the rat. They markedly increase when the newborns are weaned on a high-carbohydrate diet but not when they are weaned on a high-fat diet. Force-feeding suckling rats with carbohydrate is associated with a rapid (2 hr) increase of liver glucokinase mRNA. This suggests that hyperinsulinemia is involved in the stimulation of liver glucokinase gene transcription.

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ACKNOWLEDGMENTS

We thank Drs. A. G. Goodridge, R. W. Hanson, P. B. Iynedjian and K. H. Kim for kindly providing us with the cDNA probes used in this study, P. Maulard and B. Gouhot for technical assistance, and D. Chamereau for taking care of the animals. This work was supported by grants from the Minist~re de la Recherche et de la Technologie (no. 501247) and from Fondation pour la Recherche M6dicale, France. Michael Narkewicz was a recipient of a Fellowship from the Pediatric Scientist Training Program of the National Institute of Health (USA) (no.\HD-00850).

REFERENCES 1. S. HENNING, Postnatal development: coordination of feeding, digestion, and metabolism, Am. J. Physiol. 241, G199-G214 (1981). 2. J. GIRARD, P. FERRE, A. KERVRAN, J. P. PEGORIER and R. ASSAN, Role of the insulin/glucagon ratio in the changes of hepatic metabolism during development of the rat. pp. 563-581 in Glucagon: Its Role in Physiology and Clinical Medicine, (P. P. FOA, J. S. BAJAJ and N. L. FOA, eds.), Springer-Verlag, New York (1977). 3. F. J. BALLARD and R. W. HANSON, Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver, Biochem. J. 104, 866--871 (1967). 4. R . G . VERNON and D. G. WALKER, Gluconeogenesis from lactate in the developing rat. Studies in vivo., Biochem. J. 127, 531-537 (1972). 5. M. A. BEAUDRY, J. L. CHIASSON and J. H. EXTON, Gluconeogenesis in suckling rat, Am. J. PhysioL 233, E175-E180 (1977). 6. J. F. DECAUX, P, FERRE and J. GIRARD, Effect of weaning on different diets on hepatic gluconeogenesis in the rat, Biol. Neonate 51,331-336 (1986). 7. F . J . BALLARD and R. W. HANSON, Changes in lipid synthesis in rat liver during development, Biochem. J. 102, 952-958 (1967). 8. C. B. TAYLOR, E. BAILEY and W. BARTLEY, Changes in hepatic lipogenesis during development of the rat, Biochem. J. 105, 717-722 (1967). 9. J. R. WEBB and E. BAILEY, Developmental changes in the activities of some enzymes associated with hepatic lipogenesis in the suckling rat, Int. J. Biochem. 6, 807-811 (1975). 10. R. G. VERNON and D. G. WALKER, Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver, Biochem. J. 106, 321-329 (1968). 11. E. A. LOCKWOOD, E. BAILEY and C. B. TAYLOR, Factors involved in changes in hepatic lipogenesis during development of the rat, Biochem. J. 118, 155-162 (1970). 12. P. HAHN, Fatty-acid synthesis in brown and white adipose tissue and liver of the rat during development, Physiol. Bohemoslov. 19, 369--373 (1970). 13. D. PILLAY and E. BAILEY, Lipogenesis at the suckling-weaning transition in liver and brown adipose tissue of the rat, Biochim. Biophys. Acta 713, 663-669 (1982). 14. S. SMITH and S. ABRAHAM, Fatty acid synthesis in developing mouse liver, Arch. Biochem. Biophys. 136, 112-121 (1970). 15. J. J. VOLPE, T. O. LYLES, D. A. K. RONCARI and P. R. VAGELOS, Fatty acid synthetase of developing brain and liver. Content, synthesis, and degradation during development, J. Biol. Chem. 248, 2502-2513 (1973).

106

D. PERDEREAU, et al.

16. A. PEREZ-CASTILLO, H. L. SCHWARTZ and J. H. OPPENHEIMER, Rat hepatic mRNA-S14 and lipogenic enzymes during weaning: role of S14 in lipogenesis, Am. J. Physiol. 253, E536-E542 (1987). 17. G. GANDEMER, G. PASCAL and G. DURAND, In vivo changes in the rates of total lipid and fatty-acid synthesis in liver and white adipose tissues of male rats during postweaning growth, Int. J. Biochem. 14, 797-804 (1982). 18. D. G. WALKER and G. HOLLAND, The development of hepatic glucokinase in the neonatal rat, Biochem. J. 97, 845-854 (1965). 19. S. C. JAMDAR and O. G R E E N G A R D , Premature formation of glucokinase in developing rat liver, J. Biol. Chem. 245, 2779-2783 (1970). 20. H. YOO-WARREN, M. A. C I M B A L A , K. FELZ, J. E. MONAHAN, J. P. LEIS and R. W. HANSON, Identification of a cDNA clone to phosphoenolpyruvate carboxykinase (GTP) from rat cytosol. Alterations in phosphoenolpyruvate carboxykinase RNA levels detectable by hybridization, J. Biol. Chem. 256, 10224-10227 (1981). 21. D. H. BAI, M. E. PAPE, F. LOPEZ-CASILLAS, X. C. LUO, J. E. DIXON and K. H. KIM, Molecular cloning of cDNA for acetyl-CoA carboxylase, J. Biol. Chem. 261, 12395-12399 (1986). 22. C. YAN, E. A. WOOD and J. W. PORTER, Characterization of fatty acid synthetase cDNA clone and its mRNA, Biochem. Biophys. Res. Commun. 126, 1235-1241 (1985). 23. P. B. IYNEDJIAN, C. UCLA and B. MACH, Molecular cloning of glucokinase cDNA. Developmental and dietary regulation of glucokinase mRNA in rat liver, J. Biol. Chem. 262, 6032-6038 (1987). 24. R. S. REDMAN and L. R. SWENEY, Changes in diet and patterns of feeding activity of developing rat, J. Nutr. 106, 615-626 (1976). 25. J. GIRARD, G. S. CUENDET, E. B. MARLISS, A. KERVRAN, M. RIEUTORT and R. ASSAN, Fuels, hormones and liver metabolism at term and during the early neonatal period in the rat, J. Clin. Invest. 52, 3190-3200 (1973). 26. C. COUPE, D. P E R D E R E A U , P. FERRE, Y. HITIER, M. NARKEWICZ and J. GIRARD, Lipogenic enzyme activities and mRNA in rat adipose tissue at weaning, Am. J. Physiol. 258, in press (1990). 27. K. SCHERRER and J. DARNELL, Sedimentation characteristics of rapidly labelled RNA from Hela cells, Biochem. Biophys. Res. Commun. 7,486--490 (1962). 28. L. G. DAVIS, M. D. DIBNER and J. F. BATTEY, Basic Methods in Molecular Biology, Elsevier Science Publishing, New York (1986). 29. P. S. THOMAS, Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose, Proc. NatL Acad. Sci. U.S.A. 77, 5201-5205 (1980). 30. A. P. FEINBERG and B. VOGELSTEIN, A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity, Analyt. Biochem. 132, 6-13 (1983). 31. K. H. KIM, Regulation of acetyl-CoA carboxylase, Curr. Top. Cell. Regul. 22, 143-176 (1983). 32. S. NAKANISHI and S. NUMA, Purification of rat liver acetyl-CoA carboxylase and immunochemical studies on its synthesis and degradation, Eur. J. Biochem. 16, 161-173 (1970). 33. J . J . VOLPE and P. R. VAGELOS, Regulation of mammalian fatty acid synthetase. The roles of carbohydrate and insulin, Proc. Natl. Acad. Sci. U.S.A. 71, 889--893 (1974). 34. C. M. NEPOKROEFF, K. ADACHI, C. YAN and J. W. PORTER, Cloning of DNA complementary to rat liver fatty acid synthetase mRNA, Eur. J. Biochem. 140, 441--445 (1984). 35. M. E. PAPE, F. LOPEZ-CASILLAS and K. H. KIM, Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl-CoA carboxylase mRNA, Arch. Biochem. Biophys. 267, 104-109 (1988). 36. J. D. PAULAUSKIS and H. S. SUL, Hormonal regulation of mouse fatty acid synthase gene transcription in liver, J. Biol. Chem. 264, 574-577 (1989).

GENE EXPRESSION DURING DEVELOPMENT

107

37. A. G. GOODRIDGE, Dietary regulation of gene expression: enzymes involved in carbohydrate and lipid metabolism, A. Rev. Nutr. 7, 157-185 (1987). 38. R. G. VERNON and D. G. WALKER, Adaptative behaviour of some enzymes involved in glucose utilization and glucose formation in rat liver during the weaning period, Biochem. J. 106, 331-338 (1968). 39. J. M. STOREY and E. BAILEY, Effect of streptozotocin diabetes and insulin administration on some enzyme activities in the post-weaning rat, Enzyme 23, 382-387 (1978). 40. H. C. TOWLE and C. N. MARIASH, Regulation of hepatic gene expression by lipogenic diet and thyroid hormones, Fed. Proc. 45, 2406-2411 (1986). 41. D. A. FISHER, J. H. DUSSAULT, J. SACK and I. J. CHOPRA, Ontogenesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheep and rat, Rec. Prog. Horm. Regul. 33, 59-107 (1977). 42. S. LYONNET, C. COUPE, J. GIRARD, A. KAHN and A. MUNNICH, In vivo regulation of glycolytic and gluconeogenic enzyme gene expression in newborn rat liver, J. Clin. Invest. 81, 1682-1689 (1988). 43. S. M. TILGHAM, R. W. HANSON and F. J. BALLARD, Hormonal regulation of phosphoenolpyruvate carboxykinase (GTP) in mammalian tissues, pp. 47-91 in Gluconeogenesis: Its Regulation in Mammalian Species (R. W. HANSON and M. A. MEHLMAN, eds.), John Wiley, New York (1976). 44. M. A. CIMBALA, W. H. LAMERS, K. NELSON, J. E. MONAHAN, H. YOO-WARREN and R. W. HANSON, Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP, J. Biol. Chem. 257, 7629-7636 (1982). 45. E . G . BEALE, J. L. HARTLEY and D. K. GRANNER, N6,O2'-dibutyryl cyclic AMP and glucose regulate the amount of mRNA coding for hepatic phosphoenolpyruvate carboxykinase (GTP), J. Biol. Chem. 257, 2022-2028 (1982). 46. E . G . BEALE, T. ANDREONE, S, KOCH, M. GRANNER and D. K. GRANNER, Insulin and glucagon regulate cytosolic phosphoenolpyruvate carboxykinase (GTP) mRNA in rat liver, Diabetes 33,328-332 (1984). 47. D . K . GRANNER and T. L. ANDREONE, Insulin modulation of gene expression, Diabetes~Metabolism Reviews 1, 139-170 (1985). 48. W. H. LAMERS, R. W. HANSON and H. M. MEISNER, cAMP stimulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase in rat liver nuclei, Proc. Natl. Acad. Sci. U.S.A. 79, 5137-5141 (1982). 49. D. K. GRANNER, T. ANDREONE, K. SAZAKI and E. G. BEALE, Inhibition of transcription of the phosphoenolpyruvate carboxykinase gene by insulin, Nature 305, 549-551 (1983). 50. D . G . WALKER and S. W. EATON, Regulation of development of hepatic glucokinase in the neonatal rat by the diet, Biochem. J. 105, 771-777 (1967). 51. M. J, O. WAKELAM, C. ARAGON, C. GIMENEZ, M. B. ALLEN and D. G. WALKER, Thyroid hormones and the precocious induction of hepatic glucokinase in the neonatal rat, Eur. J. Biochem. 100, 467-475 (1979). 52. P. M. HANEY, C. R. ESTRIN, A. CALIENDO and M. S. PATEL, Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet, Arch. Biochem. Biophys. 244, 787-794 (1986). 53. M. J. O. WAKELAM and D. G. WALKER, De novo synthesis of glucokinase in hepatocytes isolated from neonatal rats, FEBS Lett. 111, 115-119 (1980). 54. T. NAKAMURA, K. AOYAMA and A. ICHIHARA, Precocious induction of glucokinase in primary cultures of postnatal rat hepatocytes, Biochem. Biophys. Res. Commun. 91,515-520 (1979). 55. M . R . NARKEWICZ, P. FERRE and J. GIRARD, Regulation of the development of glucokinase mRNA in primary culture of hepatocytes from suckling rats, Diabetologia 32,521 A (1989). 56. P. B. IYNEDJIAN, A. GJINOVCI and A. E. RENOLD, Stimulation by insulin of glucokinase gene transcription in liver of diabetic rats, J. Biol. Chem. 263, 740-744 (1988).

108

D. P E R D E R E A U , et al.

57. M. A. MAGNUSSON, T. L. ANDREONE, R. L. PRINTZ, S. KOCH and D. K. GRANNER, Rat glucokinase gene: structure and regulation by insulin, Proc. Natl. Acad. Sci. U.S.A. 86, 4838-4842 (1989). 58. T. ISSAD, C. COUPE, P. FERRE and J. GIRARD, Insulin-resistance during suckling period in rats, A m . J. Physiol. 253, E142-E148 (1987).