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Articles

Dietary Protein Concentration Regulates the mRNA Expression of Chicken Hepatic Malic Enzyme^{1 ,2}

Department of Poultry Science, University of Georgia, Athens, GA 30602

³To whom correspondence should be addressed. E-mail: ajdavis{at}arches.uga.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Chicken hepatic malic enzyme activity varies with dietary protein content. The mechanisms responsible for this alteration in activity are unclear. In a series of four experiments, broiler chicks were allowed free access for 1.5, 3, 6 or 24 h to a low (13 g/100 g diet), basal (22 g/100 g diet) or high (40 g/100 g diet) protein diet. The diets were isocaloric and had equal concentrations of dietary fat. Hepatic malic enzyme mRNA expression and enzyme activity as well as total liver lipid concentration were examined for each experimental duration. There were no differences in the expression of the mRNA for malic enzyme at 1.5 h, but at 3, 6 and 24 h, malic enzyme mRNA expression was significantly (P < 0.05) reduced in chicks fed the high protein diet and significantly enhanced in chicks fed the low protein diet compared with chicks fed the basal diet. Hepatic malic enzyme activities and total lipid concentration were not different among the chicks fed the different diets at 1.5 and 3 h. At 6 and 24 h, malic enzyme activity and total liver lipid concentration were both significantly greater in birds fed the low protein diet compared with levels in the birds fed the other two diets. In birds fed the high protein diet, malic enzyme activity and total liver lipid concentration were significantly reduced at 24 h compared with birds fed the basal diet. In a final experiment, the observed differences in malic enzyme mRNA expression at 6 h were confirmed when chicks were given access to isocaloric diets with the same protein levels as the initial 4 experiments, but with the dietary concentration of carbohydrate held constant. The results suggest that previously observed alterations in the activity of malic enzyme, which were correlated with dietary protein intake, are due to rapid changes in the mRNA expression of this enzyme.

KEY WORDS: • chickens • malic enzyme • temporal change • protein

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Malic enzyme [L-malate-NADP⁺ oxidoreductase (decarboxylating), EC 1.1.1.40] is a cytoplasmic protein that catalyzes the oxidative decarboxylation of malate to pyruvate and CO₂, while simultaneously generating NADPH from NADP⁺. The NADPH generated can be utilized in de novo synthesis of palmitate, which is the precursor molecule for the formation of other long-chain fatty acids. In avian species, the liver is the main site for the de novo synthesis of fatty acids (1 2 3) . Most of the NADPH necessary for the synthesis of fatty acids in birds is believed to be derived from the activity of malic enzyme (4 5 6) because the hepatic monophosphate-shunt dehydrogenases appear not to play an important role in chick hepatic lipogenesis (4 ,5 ,7) . The activity of hepatic malic enzyme is highly positively correlated with the rate of fatty acid synthesis, the percentage of body fat and the percentage of abdominal fat in chicks (6 ,8 9 10) .

The activity of malic enzyme is high in well-fed birds and low in starved birds (10) . In fact, the steady-state concentration of malic enzyme mRNA in refed chicks is 35 times greater than the level found in starved chicks (11) . In several subsequent experiments [as reviewed by Goodridge et al. (11 ,12) ], Goodridge and co-workers demonstrated that nutritionally and hormonally induced changes in malic enzyme activity were accompanied by comparable changes in enzyme synthesis and in the abundance of malic enzyme mRNA.

In addition to being regulated by feeding status, malic enzyme activity is regulated by dietary protein intake. In force-feeding experiments with chicks, when dietary carbohydrate and fat were kept constant while dietary protein was increased, the activity of malic enzyme and fatty acid synthesis decreased (13) . Subsequent reports have strengthened this original finding that dietary protein is an intrinsic regulator of malic enzyme activity and the synthesis of liver fatty acids in chicks (14 15 16 17 18 19 20 21 22) . The mechanisms by which dietary protein regulates chick hepatic malic enzyme activity are unclear. Therefore, the current study was conducted to determine whether the changes in chicken hepatic malic enzyme activity and liver lipid concentration related to dietary protein intake are preceded by changes in the amount of malic enzyme mRNA, and to establish the temporal response of malic enzyme activity in chicks after consumption of altered dietary protein levels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Arbor Acre mix-sexed broiler chicks (1 d old) obtained from Seaboard Farms (Athens, GA) were raised in thermostatically controlled, electrically heated battery brooder cages with wire floors. The cages were lighted for 24 h/d. Chicks had free access to water and a practical chick starter diet. Six or seven days after hatching, the chicks were sorted and those with extreme weights discarded. The remaining chicks were assigned to experimental groups so as to achieve similar weight distributions among all pens. The chicks were killed by cervical dislocation at the end of experiments to obtain liver samples. The Instructional Animal Care and Use Committee of the University of Georgia approved all animal procedures.

Experiment 1.

After sorting, the chicks were randomly assigned to 36 pens each consisting of two birds. The chicks were fed a semipurified basal diet containing 22 g protein/100 g diet (Table 1 ) for 4 d to allow them to acclimate to the semipurified diet. After this adjustment period, the 36 pens were split into three groups and the chicks were then fed the basal diet, a low protein diet (13 g/100 g diet) or a high protein diet (40 g/100 g) (Table 1) for 6 or 24 h. Therefore, there were 6 replicate pens of two birds each for the three dietary treatments at both time points. At the end of each experimental period, total feed consumption was determined for each pen. Whole livers were quickly excised from both chicks of each pen. A sample of 250 mg was taken from the left lobe of each liver and combined for RNA isolation. The remainder of the livers was then kept in an ice-cold 0.25 mol/L sucrose solution containing 1 nmol/L reduced glutathione for subsequent determination of malic enzyme activity and total liver lipid concentration.

All of the protocols were the same in this experiment as in Experiment 1, except that the chicks were fed the experimental diets for only 1.5 and 3 h.

Experiments 3 and 4.

These two experiments were conducted to confirm the mRNA results obtained in Experiments 1 and 2 and to determine total liver lipid concentration. The protocols for these two experiments followed those of Experiments 1 and 2.

Experiment 5.

This experiment was done to test whether the dietary protein–induced changes in malic enzyme mRNA expression detected in Experiments 1–4 would still occur when the concentration of dietary carbohydrate was kept constant. In Experiments 1–4, the level of dietary fat was kept constant while the levels of protein and carbohydrate changed as the levels of isolated soybean protein and glucose monohydrate were adjusted to achieve isocaloric low or high protein diets. In this experiment, the amount of dietary fat was adjusted to achieve isocaloric dietary treatments while formulating the low, basal and high protein diets. Because corn oil has more than twice the energy density of glucose monohydrate, sand had to be incorporated into some of the experimental diets to maintain equal energy concentrations per gram of experimental diet. In this experiment, we decided not to make any comparisons between the low protein and high protein diets because to make this comparison, the low protein diet would have contained >15 g corn oil/100 g and 15 g sand/100 g to keep the dietary carbohydrate levels equal in the diets. Therefore, chicks fed the basal diet were compared with chicks fed a low protein diet containing an equivalent amount of glucose monohydrate. Similarly, chicks fed the previously tested high protein diet were compared with chicks fed a new basal diet containing a level of glucose monohydrate equivalent to that of the high protein diet. The composition of the adjusted low protein and basal experimental diets is presented in Table 1 . There were six replicate pens of two birds for each dietary treatment. Six hours after access was given to the experimental diets, liver samples were collected and pooled by pen for Northern analysis of malic enzyme mRNA.

Malic enzyme assay.

After collection, livers pooled by pen were blotted dry and finely minced after removal of any connective tissue. Minced tissue (2 g) was then homogenized with 9 parts ice-cold 0.25 mol/L sucrose solution containing 1 mmol/L reduced glutathione with motor driven (990 rpm) Teflon pestles in a glass tube. Five passes were made through the tissue with a Teflon-to-smooth glass clearance of 0.06 cm followed by five passes with a pestle-to-glass clearance of 0.03 cm. The homogenate was centrifuged at 4°C for 10 min at 700 x g using a Sorvall RC-2B (Newton, CT) centrifuge. The supernatant was recovered and 8 mL of the 0.25 mol/L sucrose solution was added to the supernatant; the samples were then recentrifuged at 15,900 x g at 4°C for 10 min. The supernatant was saved and frozen at -80°C.

Frozen cytosol was thawed on ice and then centrifuged at 100,000 x g in a Beckman L8-M ultracentrifuge (Palo Alto, CA) at 4°C for 60 min. In general, the procedures for the malic enzyme assay were modified from those of Ochoa (23) . The assay was conducted at room temperature. To a 3-mL quartz cuvette, 0.01 mL of supernatant from the 100,000 x g centrifugation, 1.8 mL of buffer containing 75 mmol/L TrisHCL and 2.8 mmol/L MnCl₂, and 0.9 mL of 0.26 mmol/L NADP were added. A reading was taken on a Beckman DU530 spectrophotometer (Fullerton, CA) at 340 nm for 1 min to detect any endogenous reducing activity. To begin the reaction, 0.1 mL of 0.3 mol/L L-malate was added to the cuvette. A reading was again taken for 1 min at 340 nm. The change in optical density as NADP was converted to NADPH from 15 to 45 s after adding the malate minus the change due to endogenous activity was used for calculation of malic enzyme activity in units of nanomoles of NADP reduced per minute. The amount of activity was then corrected for the amount of protein in the sample. Protein concentration was determined using the method of Lowry et al. (24) with bovine serum albumin as a standard.

Liver lipid content.

Total liver lipid content was determined on homogenized liver samples using the method of Folch et al. (25)

RNA extraction and Northern blot analysis.

Total RNA was extracted from liver samples pooled by pen using a guanidine isothiocynate/phenol-chloroform method (26) . Total RNA (40 µg/sample) was run on an agarose/formaldehyde gel and then transferred to a nylon membrane as previously described (27) . Duck malic enzyme (28) and chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (27) cDNA clones were prepared and labeled with ³²P for Northern blot analysis as previously described (27) . The hybridization and densitometry procedures also followed those described previously (27) . For each experimental duration, there were two Northern blots with the replicate samples for each dietary treatment split evenly between the two blots. The two blots were hybridized at the same time and exposed together on the same film. Relative mRNA expression of malic enzyme was determined for the samples of each blot by calculating the signal intensity for each sample relative to the strongest malic enzyme signal, which was assigned a value of 1. Before calculation of relative malic enzyme mRNA levels, GAPDH mRNA expression was used to correct the malic enzyme values for equality of RNA loading and transfer for each blot.

Statistical analyses.

Data from each experiment were subjected to ANOVA according to the General Linear Model procedure using replicate and dietary protein levels as factors. Tukey’s multiple-comparison procedure (29) was used to detect significant differences among the diets. All statistical procedures were done with the Minitab statistical software package (Release 8.2, State College, PA), and differences were considered significant when P-values were <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Experiment 1.

For the 6-h experimental period, food consumption was 21 ± 0.5, 20 ± 2 and 15 ± 2 g/chick for the low protein, basal and high protein diets, respectively. Food consumption over the 24-h experimental period was 22 ± 3, 21 ± 1 and 18 ± 2 g/chick for the low protein, basal and high protein diets, respectively. The only significant difference (P < 0.05) in food consumption was between the chicks fed the low protein diet and those fed the high protein diet for 6 h.

Expression of malic enzyme mRNA was significantly different among the three diet groups at both 6 and 24 h with expression decreasing as dietary protein increased (Fig. 1A ). The differences among groups in malic enzyme activity at 24 h mirrored those of the mRNA (Fig. 1 B). At 6 h, however, malic enzyme activity did not differ between the chicks fed the basal and high protein diets, whereas that in chicks fed the low protein diet was significantly greater than in the other two diets.

View larger version (36K): [in this window] [in a new window]   Figure 1. The relative density of hepatic malic enzyme mRNA (A) and malic enzyme activity of liver homogenates (B) of chicks fed different dietary protein concentrations [Experiments 1 (6 and 24 h) and 2 (1.5 and 3 h)]. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05. Note that the relative densities of malic enzyme mRNA to one another are specific for each time point and that all statistical comparisons are within a given time period.

Food consumption did not differ among the chicks fed the three different experimental diets for 1.5 or 3 h. For the 1.5-h experimental period, food consumption was 2 ± 0.5, 3 ± 0.5 and 2 ± 0.5 g/chick, whereas for the 3-h experimental period, consumption was 7 ± 1, 9 ± 1 and 9 ± 2 g/chick for the low protein, basal and high protein diets, respectively.

Expression of malic enzyme mRNA was not significantly different among the chicks fed all three diets at 1.5 h, but it did differ among the three groups at 3 h (Fig. 1 A). There were no differences in hepatic malic enzyme activity among the three groups at either 1.5 or 3 h (Fig. 1 B).

Experiment 3.

Food consumption of the chicks fed the low protein, basal and high protein diets over the 6-h experimental period was 13 ± 1, 16 ± 1 and 15 ± 3, g/chick, respectively, and 21 ± 0.5, 23 ± 1 and 20 ± 1 g/chick, respectively, for the 24-h experimental period. There were no significant differences at either time point in food consumption among the chicks fed the three diets.

Differences in malic enzyme mRNA expression were the same as in Experiment 1, with expression of malic enzyme mRNA increasing as the dietary protein level decreased. Chick liver total lipid concentration at 24 h was significantly different among the three diet groups with total lipid concentration increasing as the dietary protein level decreased (Fig. 2 ). At 6 h, total liver lipid concentration in the livers of birds fed the low protein diet was greater than those of birds fed the other two protein levels.

View larger version (50K): [in this window] [in a new window]   Figure 2. Liver total lipid concentration in chicks fed different dietary protein concentrations [Experiments 3 (6 and 24 h) and 4 (1.5 and 3 h)]. Values are means ± SEM, n = 6 replicate pens. Means at a time with different letters differ, P < 0.05.

Food consumption for the chicks fed the diets for 1.5 and 3 h were not different. Food consumption for the chicks fed the low protein, basal and high protein diets over the 1.5-h experimental period was 5 ± 0.5, 5 ± 0.1 and 4 ± 0.6 g/chick, respectively; over the 3-h experimental period, it was 8 ± 1, 9 ± 0.5 and 7 ± 1 g/chick, respectively. Liver total lipid concentration was not significantly different for the chicks fed the three experimental diets for either 1.5 or 3 h (Fig. 2) .

The expression pattern of the mRNA for malic enzyme in this experiment was the same as it was for Experiment 2. Representative Northern blots from Experiments 3 and 4 are shown in Figure 3 .

View larger version (75K): [in this window] [in a new window]   Figure 3. Autoradiograms from the Northern analysis of liver malic enzyme, showing 3 of the 6 replicate samples from each dietary treatment for the 1.5-, 3-, 6- and 24-h experimental durations [Experiments 3 (6 and 24 h) and 4 (1.5 and 3 h)]. Total RNA (40 µg) was loaded for each sample. Samples obtained from birds fed the low protein, basal and high protein diets are in lanes 1–3, 4–6 and 7–9, respectively. The film exposure times were 44, 56, 72 and 72 h for the malic enzyme Northern blots from the 24-, 6-, 3- and 1.5-h experimental times, respectively. GAPDH films were all exposed for 1.5 h. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ME, malic enzyme.

Food consumption over the 6-h experimental period did not differ among groups and was 9 ± 1, 9 ± 0.3, 9 ± 1 and 8 ± 0.3 g/chick for the adjusted low protein, original basal, adjusted basal and original high protein diets, respectively.

When dietary carbohydrate was maintained as dietary protein was altered, expression of hepatic malic enzyme mRNA was still significantly enhanced in chicks fed the low protein diet compared with those fed the basal diet (Fig. 4 ). Furthermore, expression of hepatic malic enzyme mRNA was significantly reduced in chicks fed the high protein diet compared with chicks fed the adjusted basal diet with an equivalent carbohydrate level.

View larger version (47K): [in this window] [in a new window]   Figure 4. The relative density of hepatic malic enzyme mRNA in chicks fed the four dietary treatments (Experiment 5). Values are means ± SEM, n = 6 replicate pens. Means with different letters differ, P < 0.05. The adjusted low protein and basal diets had equal carbohydrate levels as did the adjusted basal and high protein diets.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In contrast, chicks switched to a high protein diet had a decrease in the expression of hepatic malic enzyme mRNA after 3 h. The decrease in malic enzyme mRNA was followed by a decrease in malic enzyme activity and hepatic lipid concentration 24 h after feeding the high protein diet. The reason for the delayed decrease in malic enzyme activity in the liver of chicks fed the high protein diet compared with the more rapid response observed with the low protein diet is unclear, but may indicate that the response to the high protein diet involves more than simply changes in malic enzyme mRNA concentration.

Interestingly, Rosebrough et al. (21) also reported differences in the response time of malic enzyme activity when chicks were fed either a high (21 g/100 g diet) or low (12 g/100 g diet) protein diet for 21 d and then switched to the opposite diet. Chicks fed the low protein diet and then switched to the high protein diet had a significant decrease in malic enzyme activity when measured for the first time 2 d after the dietary switch. In contrast, those chicks fed the high protein diet and then switched to a low protein diet did not have a significant increase in hepatic malic enzyme activity until the next measurement was taken 5 d after the dietary switch.

In designing dietary experiments to measure malic enzyme activity, there is a need to keep energy intake equal in the different dietary treatments because malic enzyme activity and fatty acid synthesis are sensitive to total energy intake (11 ,30) . Given that the duration of our experiments was short and feed intake was expected to be similar among the treatment groups, the experimental diets were designed to be isocaloric. In the first four experiments, the level of dietary fat was held constant while the level of dietary protein was altered, but dietary carbohydrate also was changed to keep the diets isocaloric. Dietary fat was kept constant because consumption of increased levels of dietary fat has been associated with decreased fat synthesis (13 ,31 32 33 34) . It could be argued, however, that the observed changes in malic enzyme mRNA expression and activity in Experiments 1–4 were due entirely to alterations in dietary carbohydrate and not related to dietary protein intake.

To better establish the effect of dietary protein on malic enzyme, the level of carbohydrate was held constant in Experiment 5 as dietary protein and fat changed. As was found in the initial experiments, in this experiment, malic enzyme mRNA levels were increased after feeding the low protein diet for 6 h and decreased after feeding the high protein diet for 6 h. Again, it could be argued that these results were related to dietary fat content instead of dietary protein content. Given that the responses were similar to those of the previous experiments, however, this seems unlikely. Furthermore, increasing dietary fat is associated with reduced fatty acid synthesis (13 ,32 33) . In this experiment, however, a low protein, high fat diet increased malic enzyme mRNA, indicating that dietary protein has a very specific and strong influence on malic enzyme activity. This is a conclusion supported by several previous reports (14 15 16 17 18 19 20 21 22) .

Hillard et al. (35) reported that the inhibitory influence of dietary fat on chick liver fatty acid synthesis was not a direct action of fat per se, but was secondary to a reduced carbohydrate intake that resulted as dietary fat replaced dietary carbohydrate. This would suggest that the observed tendency (P = 0.089) for chicks fed the "adjusted" basal diet to have a lower expression of malic enzyme mRNA than chicks fed the original basal diet (Fig. 4) was due to the lower carbohydrate content of this diet.

As has been found with avian species, the activity of malic enzyme in fish (36) and rats (37 38 39 40 41 42) also decreases with increasing dietary protein intake. There is some indication that the dietary protein effect on malic enzyme activity in rats may be generated in part by the intake of specific dietary amino acid profiles or specific amino acids (39 ,41) . The hepatic expression of malic enzyme mRNA was examined by Katsurada et al. (43 ,44) in rats deprived of food for 2 d and then refed diets containing 0, 18 or 85 g of protein/100 g diet. When first examined 12 h after the initiation of refeeding, malic enzyme mRNA concentrations were significantly lower in the rats fed the highest level of protein compared with those fed the protein-free diet. The activity of malic enzyme was also different between these two dietary groups at 12 h, but the differences became even more pronounced at 24 and 48 h. Although malic enzyme mRNA concentrations were not different between the rats refed diets containing 0 and 18 g protein/100 g diet, malic enzyme activity was consistently and significantly lower in the rats refed the protein-free diet. On the basis of this finding, Katsurada et al. (43 ,44) suggested that dietary protein may also regulate translation of malic enzyme.

The mechanism by which dietary protein concentration regulates the expression of malic enzyme mRNA is unclear. Research is warranted to determine whether the concentrations of specific amino acids are responsible for the observed protein effect and to determine the identity of the regulatory molecules that mediate the response of malic enzyme mRNA expression to changes in dietary protein concentration. Finally, our data, like previous reports (6 ,10) , indicate that the activity of malic enzyme, which provides the necessary NADPH for lipogenesis, is correlated with de novo lipogenesis. Yeh and Leveille (10) and Tanaka et al. (13) suggested that the availability of NADPH regulates lipogenesis in chicks fed high protein diets. In contrast, Goodridge (45) and Rosebrough et al. (20) suggested that the activity of malic enzyme is more a function of NADPH utilization. Given the rapid responses of malic enzyme seen in the present work, it may be more likely that the activities of malic enzyme and the other enzymes involved in fatty acid synthesis in chicks are simply regulated in concert.

In summary, switching chicks from a basal diet to a low or high protein diet resulted in a rapid (3 h) change in the expression of the mRNA for malic enzyme. A switch to a low protein diet increased the level of malic enzyme mRNA, whereas feeding a high protein diet decreased its level. The changes in malic enzyme mRNA were associated with subsequent changes in malic enzyme activity and liver total lipid concentration. The results indicate that dietary protein per se is a regulator of malic enzyme synthesis.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the generous gifts of the duck malic enzyme cDNA clone from Susan Stapleton of Western Michigan University and of the chicken GAPDH cDNA clone from Patricia Johnson of Cornell University.

	FOOTNOTES

 
¹ Presented in part at the 89th Annual Poultry Science Association Meeting, August 2000, Montreal, Canada [Adams, K. A. & Davis, A. J. (2000) Dietary protein level regulates the mRNA expression of chicken hepatic malic enzyme. Poult. Sci. 79 (suppl. 1): A178 (abs.)].

² Supported in part by Hatch Project GEO00865.

Manuscript received March 27, 2001. Initial review completed May 14, 2001. Revision accepted June 26, 2001.

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