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Metabolic effects of high-protein diets in Zucker rats
Metabolic
Effects
of High-Protein
Diets in Zucker Rats
Jean Peret, And& C. Bach, Brigitte Delhomme, Brigitte Bois-Joyeux, Marc Chanez, and Henri Schirardin The effects of dietary protein on the metabolism of proteins, carbohydrates, and especially, lipids were investigated in genetically obese Zucker rats and their lean siblings. For 46 days the rats received diets containing 15%. 64%. or 82% protein, included at the expense of cornstarch. In the obese animals, the high-protein diets led to decreased food intake and weight gain. While these diets decreased the activities of lipogenic enzymes along with the lipid gain, they did not decrease the final body-fat content. The increased protein intake stimulated hepatic ureogenesis and gluconeogenesis. Lipolysis was stimulated, as demonstrated by an accumulation of ketone bodies in the liver. Blood levels of triacylglycerols, free glycerol, and nonesterified fatty acids were concomitantly decreased, which suggests an accelerated turnover of lipids. Whatever the composition of the diet, total energy retention of the lean rats was always less than that of the obese rats. The changes observed on high-protein diets were essentially the same for the two groups, except that the final body-content of lipids in the lean rats was significantly lower. In the absence of exogenous carbohydrate, the lean rats were barely able to retain nitrogen and to maintain hepatic lipogenesis. Unlike the rats from other strains, the lean Zucker rats could not adapt to a low-carbohydrate diet: this failure may be due to a metabolic disorder.
EVERAL
STUDIES
have shown that the effi-
S ciency of energy storage is higher in obese Zucker
rats (fa/fa) than in their lean littermates (Fa/-),lm4 and that the hyperphagia of fa/fa rats5’6 is a major factor determining the deposition of excess lipid. However, neither pair-feeding of obese fatty rats to the food intake of lean anima1s,‘-337 or restriction of food intake,’ or prolonged fasting’ leads to a normalization of body-fat content or elimination of other defects in these animals.” For the past decade,” the use of high-protein diets has been advocated for the treatment of human obesity. Replacing carbohydrates with proteins in the diet of Wistar rats decreases their weight gain, body lipids, and hepatic 1ipogenesis.‘2-‘4 However, not all authors regard the use of low-carbohydrate, high-protein diet as desirab1e.‘5,‘6 Therefore, this study on the effect of high dietary protein on the deposition of fat in the genetically obese Zucker rat was initiated.” Two highprotein diets were examined; one provided some carbohydrates and the other was devoid of carbohydrates. The results obtained showed that the high-protein diets lead to decreased food intake and weight gain, but they did not decrease the final body-fat content. MATERIALS AND METHODS
Corporation (Cleveland, OH, USA). Cornstarch was obtained from Roth (Karlsruhe, West Germany) and sunflower oil from the Societe des Produits Bertrand (Crigny, France). Sodium pentobarbital (Nembutal) was from Abbott (Saint-R&my-sur-Avre, France). Plasma-free glycerol and triacylglycerol, phospholipids, and total cholesterol were determined with analysis kits as follows: triglycerides, Merckotest (E. Merck, Darmstadt, West Germany); phospholipids B-test (Wako Pure Chemical Industries, Osaka, Japan); cholesterol C-test (Wako Pure Chemical Industries, Osaka, Japan). The other enzymatic reagents were from Boehringer (Mannheim, West Germany).
Animals The lean (Fa/-) and obese (fa/fa) male Zucker rats were obtained at 6 weeks of age from CSEAL-CNRS (Orleans-la-Source, France). They were kept in individual Makrolon cages in a room at 22°C + 2OC, with a 12/ 12 h light/dark cycle (lights on at 7 AM).
Experimental
Design
The rats were fed a standard UP-HC diet (Table 1). After a period of adaptation of eight days they were distributed into three groups of obese and three groups of lean rats. Each group consisting of eight rats received one of the diets described in Table I. We refer to the diets as UP-HC (usual-protein, high-carbohydrate), HP-LC (high-protein, low-carbohydrate) and HP-CF (high-protein, carbohydrate-free). Food and tap water were provided ad libitum for 40 days. During this period the weight and daily food intakes of each rat were recorded.
Materials
Sample Collection
Salt mixture,” vitamin mixture,” cellulose non-nutritive bulk, and casein were purchased from the United States Biochemical
At the end of the experimental period and during three days, one obese and one lean animal from each group were anesthetized with sodium pentobarbital (6 mg per 100 g of body weight) and killed, between 9 AM and lo:30 AM. A small sample of liver was taken by freeze-clamping. Blood was taken from the inferior vena cava in a heparinized syringe. Then other samples of liver were taken and frozen. The carcass, without blood and liver, was weighed and frozen for analysis of the final body composition. Before the start of the experiments the initial body compositions of eight young obese and eight young lean rats were determined.
From the Centre de Recherches sur la Nutrition du CNRS. Meudon-Bellevue, France, and the Laboratoire de Pathologie Generate de la Clinique Medicate A, Hbpital Civil. Strasbourg, France. Supported in part by a grant from the Instiiut National de la Sante et de la Recherche Medicate (grant N” 81.7002). Address reprint requests to Dr. Jean Peret. Cenire de Recherches sur la Nutrition du CNRS, 9 rue Jules Hetzel, 92190 MeudonBellwue, France. 0 1984 by Grune & Stratton, Inc. 0026-0495/84/3303-0002$01.00/0
200
Analytical
Methods
Glucose,*o nonesterified fatty acids?’ glycerol,2’ triacylglycerols,” phospholipids.24 and total cholesterolz5 were estimated in the plasma.
Metabolism, Vol 33, No 3
(March),
1984
HIGH-PROTEIN
DIETS IN ZUCKER RATS
201
Table 1, Composition
of Diets
UP-HC
Tukey-Hartley-Keuls
HP-LC
Diet
HP-CF
g/loo!3
RESULTS
Casein
16.3
69.4
88.8
Cornstarch
72.5
19.4
0
Sunflower oil
3.5
3.5
3.5
Salt mixture
4.3
4.3
4.3
Vitamin mixture
1.7
1.7
1.7
Cellulose non-nutritive bulk
7.7
1.7
1.7
Energy Intake; Body Weight Gain
With increased protein content in the diet obese rats reduced their food intakes (HP-LC -24%, HPCF -35%, compared with UP-HC) (Table 2). Despite the decrease in food ingested, the daily protein intake increased considerably. This results in a reduced rate of weight gain relative to the UP-HC rats (-29% and - 53%, respectively). On all the diets the lean rats ate less and gained less weight than the obese rats. Compared to lean rats fed the usual diet, those on the high-protein diets had reduced food intake (-40% and -45%, respectively), increased protein intake, and reduced daily weight gain.
kJ/lOO g diet Energy value
1596.2
1525.9
1499.1
% metabolizable energy Protein
15.7
70.1
91.2
Carbohydrate
76.0
21.3
0
8.3
8.6
Fat
8.8
UP, usual protein; HP, high protein: HC, high carbohydrate; LC, low carbohydrate: CF. carbohydrate-free.
Casein was “vitamin-free”
as it
was 92% protein. The diets contained 15% (UP-HC), 64% (HP-LC). and 82%
(HP-CF) protein. Energy values of protein, starch, and oil were
assumed to be 16.74, @-hydroxybutyrate, tamine,
urea,
16.74,
and 37.66
acetoacetate,
inorganic
kJ respectively.
lactate,
phosphate,
pyruvate,
ATP,
ADP,
Body Composition;
and AMP
Water
(by lyophilisation),
portions of liver.
methanol
(2/l,
v/v),
total cholesterol”
protein,”
Lipids
were
Whereas in the obese rats the amount of protein in the food ingested increased from 15% to 82%, the energy retention decreased by 43%, the nitrogen retention decreased by 78%, and the daily lipid gain decreased by 39% (Table 2). In terms of final body composition, the effect of the dietary protein content was so slight that none of the changes in the percentages of water, protein, or lipid were significant. As decribed previously,2,33 the carcasses of the lean rats contained more water, more protein, and less lipid per 100 g than those of the obese rats (Table 2). In the lean rats, the I-II’-CF diet led to a decrease in body lipid content (-33% compared with HP-HC). The
and lipid were estimated on
were extracted
and triacylglycerols.23
enolpyruvate
with chloroform/
phospholipids,24 and
were determined.
The hepatic activities of mafic enzyme (EC carboxykinase
(EC
4.1 .1.32),*9
I .l. l.40)?’
phospho-
and acetyl-CoA
car-
boxylase, (EC 6.4. I .2)” were measured at 37°C. The activities are expressed per milligram of cytosol protein, or per total liver. The content of water, total lipid, and protein were determined the rat carcasses according to the method of Abraham energy retention,
nitrogen
retention,
in
et al.” The
and lipid gain were derived
from the differences between the initial and final body compositions and were expressed per day of the experiment. Statistical
analysis of results (SEM,
Table 2.
analysis of variance,
Effect of Dietary Protein on Weight
and
Gain, Energy Intake, Final Body Composition. Nitrogen,
lean
224.5
Weight gain (g/d) Final body Composition (g/ 100 9) Energy retention (kJ/d) Nitrogen retention (mg/d) Lipid gain (g/day)
* 6.8”,**
2.6 + O.l”** (
Water protein (N x 6.25) llpid
67.7
* 0.5*’
19.2 +_0.5-9 7.0 + 0.3*,** 20.3
r 1.2’”
75 + 6’ 0.22
f 0.02”‘f’
Retention
of Body Energy and
end Lipid Gain
UP-HC
Energy intake (kJ/d)
Energy and Nitrogen Retention;
Lipid Gain
gfycogen, glu-
measured’” in liver samples taken by freeze-clamping. other
test) was carried out according to Snedecor
and Cochran.”
HP4C obese
354.8
t 5.6”
4.5 t 0.3” 38.7
f 0.8
15.8 1 0.5
lean
134.4
t 5.gb.**
0.7 r o.2b,” 69.2
* 0.8.’
18.0 c 0.7’
HP-CF obese
269.0
_t 9.2”
3.2 + 0.2” 37.7
lean
123.5
obese
* 5.7b.‘r
0.1 + 0.2’,.
232.1 l
-t 11.7”
2.1 + 0.2’
t 1.1
66.9
A 0.9**
37.9
15.8 i 0.4
22.4
A 1.1”
14.9 * 0.4
+ 1.0
41.5
r 1.3
6.8 + 0.5’**
44.7
? 1.7
4.7 i 0.5b.‘f
42.6
+ 1.4
100.1
f 5.8”
6.9 ? 1.6b.*’
85.7
2 6.2”
1.1 * 2.0c,**
56.5
t 2.8’
93 + 9’ 2.31
+ 0.15^
23 * qb+’ 0.11
+ 0.03b.*”
62 + 8” 2.03
* 0.15”
1 + 6’,** + 0.03’~“’
0.02
20 + 3’ 1.44 + 0.08Y
Results are expressed as means & SEM for (8) rats fed each diet for 40 days. Initial body weight, body energy, body nitrogen, and body lipid averaged 195 + 6 g, 3262
r 142 kJ, 5.7 + 0.1 g, and 64.2 + 3.2 g. respectively for obese rats and 142 + 4 g, 967 * 43 kJ, 4.6 ? 0.2 g, and 7.4 + 0.5 gfor lean rats. UP-HC. 15% protein; HP-LC. 64% protein; HP-CF, 82% protein. Statistical significance: between lean and obese, “P < 0.05, l*P < 0.01 (analysis of variance): between diets, means not followed by the same superscript letter are significantly different (Test Tukey-Hartley-Keuls) (P < 0.05, a, b, and c (in order of decreasing magnitude) in the case of lean rats and x, y, and z in the case of obese rats.
202
PERET ET AL
daily lipid gain of lean rats was not only less than that of the obese rats, but it was also reduced by half with the HP-LC diet, as compared with the UP-HC diet, and was reduced to zero with the HP-CF diet. The total energy retention and nitrogen retention were also lower in lean rats than in obese rats, and they were zero with the HP-CF diet. Close examination of the data with the HP-CF diet shows that some lean rats lost weight and at the end of the experiments had slightly negative values for energy retention, nitrogen retention, and lipid gain, while others gained a little weight and accumulated some nitrogen and fat. These differences would be explained by a difference in the genotype. The lean rats were not identified as Fa/Fa homozygotes or Fa/fa heterozygotes; the effect of a high-protein, low-carbohydrate diet may differ slightly between these two types. Plasma Glucose and Lipids Plasma glucose was similar in lean and obese rats on each diet (Fig. 1).
In obese rats, HP-LC and HP-CF diets produced a decrease in plasma triacylglycerol ( -43% and - 40%, respectively), free glycerol (- 21% and - 3.5%,), and nonesterified fatty acids (-24% and -45%), and a slight increase in blood cholesterol (+ 10% and + 24%) when compared with the UP-HC diet. The levels of triacylglycerol, free glycerol, cholesterol, and phosphohpids were significantly lower in the lean rats than in the obese rats.34 The values in lean rats were marginally affected by the composition of the diet. Liver Weight
and Liver Composition
The hepatomegaly of the obese rats was not reduced by high-protein diets (Fig. 2). The weight of liver per 100 g of body weight, although higher in obese than in lean rats, did not vary with the composition of the diet (data not shown), both in fa/fa or in Fa/rats. Therefore, a gram of liver can be considered as a metabolic unit, which makes simple the comparison 61l r
3
5,
i
OL
I
z
E
,
Y
250.
0
1
1
L
64
, a2
IS
PROTCIN
LEVEL
1
L
64
4 a2
~/lOOgd~et
Fig. 1. Effect of dietary protein on plasma glucose and lipids. Results are expressed as mean k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Triacylglycerol and phospholipid levels were calculated using average molecular weights of 885 and 801, respectively. Nonesterified fatty acids (NEFA) (expressed as pelmitic acid equivalents). Open squares, lean; closed squares. obese. UP-HC. 15% protein: HP-LC. 84% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
I
I
15
64 PROTEIN
a2 LEVEL
15 g/100,
I
64
a2
d,et
Fig. 2. Effect of dietary protein on liver weight, glycogen, and lipids. Results are expressed as means k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares, lean; closed squares, obese. Glycogen and lipids are reported as per gram wet liver. UP-HC. 15% protein; HP-LC. 84% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
HIGH-PROTEINDIETS IN ZUCKER RATS
203
between the hepatic parameters of the two types of rat. Lipid (Fig. 2) and protein (data not shown) compositions of the liver did not change in obese or lean rats in response to high-protein diets. The concentration of glycogen decreased less in obese rats (-26% with HP-LC, and -38% with HP-CF, in comparison with UP-HC) than the lean rats (- 38% and -48%, respectively). Hepatic Metabolites
High-protein diets did not produce any significant change in hepatic lactate or pyruvate concentrations both in obese or lean rats (Fig. 3). The decrease in lactate/pyruvate ratio in obese rats receiving high protein diets was not statistically significant. High protein diets produced an increase in hepatic concentration of ,&hydroxybutyrate (+ 194% with HPLC, +23 1% with HP-CF, in comparison with UP-HC) in obese rats without a parallel change in acetoacetate
(Fig. 3). In contrast, the change in hepatic P-hydroxybutyrate concentration was less marked in lean rats ( + 126% and + 132%, respectively) and was accompanied by an increase in the concentration of acetoacetate (+ 102% and +94%, respectively). The increase in total ketone bodies @I-hydroxybutyrate and acetoacetate) was of the same magnitude both in lean and obese rats (+9 1% and + 121% in the obese rats for HP-LC and HP-CF, respectively, compared with + 117% and + 119% in the lean rats). In contrast, the ratio of a-hydroxybutyrate to acetoacetate was affected differently by high protein diet in obese and lean rats: this ratio tripled in the obese rats receiving a high-protein diet, while it remained unchanged in the lean rats in the same nutritional conditions. In parallel with the increased protein intake, an
x
;il: x
a**
**b
Y
c 1 ! 1750_
*c
f
6_
3.
b
Y
I
15
!
64 PROTEIN
82
0
I
15
I
64
82
LEVELg/lO0~d~~~
Fig. 3. Effect of dietary protein on liver metabolites. Results are expressed as mean k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares, lean; closed squares. obese. The metabolites are reported as per gram wet liver. UP-HC. 15% protein; HP-LC, 64% protein; HP-CF. 82% protein. @-OH& @-hydroxybutyrate; AcAc, acetoacetate. For statistical analysis see legend of Table 2.
1
64
15 PROTEIN
LEVEL
82
g/1009 d,e,
Fig. 4. Effect of dietary protein on protein intake and on liver urea and glutamine. Results are expressed as mean f SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares. lean; closed squares, obese. Urea and glutamine are reported as per gram wet liver. UP-HC. 15% protein: HP-LC. 54% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
204
PERET ET AL
Table 3. Effect of Dietary Protein on Liver Adenine Nucleotides, Inorganic Phosphate (Pi), and Phosphorylation State Metabolites(nmol/g) UP-W
Metabolites (nmollg) ATP
2.142
f 74
745 + 65b
AMP
165 f 18b
ATP ADPMPiM-’
HP-CF
lean
obese
ADP Pi
HP-CC
lean
obese
lean
obese
1,738
+ 100
1,924
+ 93
1,759
+ 126
2.071
f 82
1,759
r 111
1.007
f 126
1,089
k 55”
1,109
+ 63
1,171
+ 64”
1,029
+ 48
177 ) 26’
265 + 29”
1,910
f 125h
1,892
t 168’
1,626
f 180
1,108
+ 207”
2,191
343 + 459
r 173”’
3.198
662 + 80b
260 + 32’
i 291”
2.787
548 + 83”
289 + 54w
i 202’
2,815
684 + 8gb
2 126’
606 f 72”
Results are expressed as means for eight rats fed each diet for 40 days. UP-HC, 15% protein; HP-LC, 64% protein; and HP-CF. 82% protein. For statistical analysis see legend of Table 2.
accumulation of urea was observed in the liver (Fig. 4).35 It is noteworthy that the lean rats accumulated as much as the obese rats, even though lean rats ingested only half the amount of protein as obese rats. The decrease in liver glutamine content was also identical in the two phenotypes. Hepatic Adenine Nucleotides Hepatic concentrations of adenine nucleotides were the same for the obese as for the lean rats (Table 3). The high-protein diets increased the AMP and inorganic phosphate content in both groups of animals and the ADP content in the lean rats. In addition, when the dietary level of protein increased, the state of phosphorylation decreased. Activities of Malic Enzyme, Acetyl-CoA and Phosphoenolpyruvate
Carboxylase,
Carboxykinase
In the obese rats, the high-protein diets produced a decrease in the activity of acetyl-CoA carboxylase and of malic enzyme, and a rise in the activity of phosphoenolpyruvate carboxykinase (Table 4). The activities of acetyl-CoA carboxylase and malic enzyme were lower in lean than in obese rats. In the lean rats the high-protein diets increased the activity of phosphoenolpyruvate carboxykinase and strongly reduced that of acetyl-CoA carboxylase.
DISCUSSION Obese Rats
The high activities of malic enzyme and of acetylCoA carboxylase (Table 4) in the liver of obese rats receiving the UP-HC diet suggest that lipid synthesis is greatly accelerated in the liver? and, also, in the adipose tissue3’ of these animals. This results in an accumulation of triacylglycerol in the liver (Fig. 2), in the carcass, (Table 2), and in the blood (Fig. 1). When the carbohydrate content of the diet was reduced (HP-LC diet) or eliminated (HP-CF diet) and replaced with proteins, it would have been logical to expect that both lipogenesis and accumulation of fat would have been reduced since carbohydrates are good lipogenic precursors. We also observed that highprotein diets decreased the daily weight gains (Table 2). Munro3* attributed this decrease to the inability of high-protein diets to ensure an adequate energy supply * This reduction of weight gain can be explained by the decrease in energy intake following the passage from a normal-protein diet to a high-protein diet (Table 2). The HP-CF diet appeared to be particularly effective; it reduced the food intake by about a third and the daily weight gain by half. Nevertheless, these animals remained obese at the end of the experiments, although they were less fatty than those receiving the UP-HC diet (Table 2). In fact, the daily lipid gain was
Table 4. Effect of Dietary Protein on Hepatic Enzyme Activities HP-LC
UP-HC lean
Enrvmes
ACC ME PEPCK
pmolltotal liver
11.8 + 2.9’+*
30.6
-e 3.4”
21.3
f 2.3’
HP-CF
lean
obese
5.5 t l.gb,** 7.5 + 2.3b.**
lean
obese
21.0
+ 2.3”
13.1 * 0.7*
4.3 + O.gb.*+ 5.9 k l.ob.+*
obese
18.2 zk 3.6”
nmol/mg proteins
18.3 + 2.9”“*
fimolltotal liver
35.9
k 2.5b”
214.4
f 18.8”
52.1
+ 2.8”.”
153.4
* 10.6”
33.5
f 3.gb.**
151.9
12.1 + 1.9” k 8.3”
nmol/mg proteins
59.8
r 5.0**
146.2
k 10.9’
66.3
+ 7.6+*
101.6
t 7.2’
57.2
+ 3.1”
101.6
+ 5.5”
pmol/total liver
11.3 + l.lb**
17.8 k 1.2”
26.0
k 2.4”
28.2
+ 3.3”
25.1
+ 3.0”
37.0
t 6.5’
nmoljmg proteins
17.1 + 1.5b
15.7 * 0.8’
40.9
i- 3.01,+*
21.1
t 1.9’
44.3
+ 3.1a**
28.2
+ 3.9’
Results are expressed as means -c SEM for eight rats fed each diet for 40 days. ACC, acetyl CoA carboxylase; ME, malic enzyme: PEPCK, phosphcenolpyruvate carboxyidnase. UP-HC. 15% protein: HP-LC, 64% protein: HP-CF. 82% protein.
For statistical
analysis see legend of Table 2.
HIGH-PROTEIN
DIETS IN ZUCKER RATS
reduced at best by a third (with the HP-CF diet), and the activities of the two enzymes involved in hepatic lipogenesis, acetyl-CoA carboxylase and malic enzyme, were also reduced by about a third (Table 4). The final body composition was the same whatever the protein content of the diet. Radcliffe and Webster39 and Jenkins and Hershberger4’ have previously reported that a high-protein diet is unable to prevent the accumulation of fat in obese Zucker rats. One wonders whether these metabolic changes are due to altered protein and carbohydrate content of the diet or to the changes in total energy intake. When obese rats on high-carbohydrate diets are pair-fed with lean rats, they still gain excess fat, for review see Bray and York.4’ Moreover, Radcliffe and Webster,42 by comparing gains in protein, lipid, and energy in obese and lean male rats pair-fed containing 10% and 50% protein, showed that the greater rate of lipid deposition in obese rats was not due to hyperphagia per se, but to an abnormal partition of metabolic energy between fat (too much) and protein (too little). Thesedata strongly suggest that the metabolic changes observed in the present study can be related to altered protein and carbohydrate composition of the diet and to the metabolic defects of obese rats. The inability of high-protein diets to reduce the percentage of fat in the carcass lies in two observations. The first, made by Radcliffe and Webster,39 is that whatever the level of protein in the diet, the obese rats maintained highly efficient energy metabolism. This is seen in the case of the HP-CF diet, with 57 kJ of energy retention per day (Table 2). Dunn and Hartsook showed by the use of radioactive tracers that the obese Zucker rat can incorporate a larger proportion of dietary amino acids in their body lipids than can the lean rats. Lipogenesis from amino acids requires that amino acids were first deaminated. Our data show that the increase in ingested proteins in obese rats is accompanied both by an increase in liver urea (Fig. 4)44 and by a decrease in nitrogen retention (Table 2). The concomitant decrease in liver glutamine in the rats receiving the high-protein diets suggests a use of this amino acid in the gluconeogenesis pathway after it has been deaminated. An increase in gluconeogenesis is essential for an organism that receives little or no exogenous carbohydrate. The extent of this stimulation is demonstrated by the increased activity of liver phosphoenolpyruvate carboxykinase (Table 4). It appears that the rise in gluconeogenesis enables the maintenance of a normal blood glucose level (Fig. 1) without preventing the fall in hepatic glycogen concentration (Fig. 2). The rates of ureogenesis and gluconeogenesis are
205
increased by high-protein diets whereas glycolysis is decreased.45 This could explain the increase in the hepatic levels of ADP, AMP, and inorganic phosphate and hence the decreased phosphorylation ratio observed in HP-LC and HP-CF rats (Table 3).35 As previously reported for Wistar rats,35l45 highprotein diets are ketogenic in Zucker rats. In this study hepatic @hydroxybutyrate tripled (Fig. 3). The increase in ketogenesis involves an acceleration of tissue lipolysis. The fact that blood triacylglycerols, free glycerol, and nonesterified fatty acids (Fig. 1) were concomitantly decreased can be explained only by an accelerated turnover of lipids. The cholesterol response was different. In the obese rats the diets poor in carbohydrates, and also in lipids, led to an increased level of blood cholesterol. We think that a fraction of the acetyl-CoA released by lipid catabolism is prevented from being totally oxidized in the Krebs cycle because oxaloacetate is decreased due to gluconeogenesis. This acetyl-CoA may contribute both to stimulating ketogenesis, as discussed above, and to increasing the synthesis, and hence, the blood level, of cholesterol. These losses are not enough, however, to reduce the lipid content of the liver (Fig. 2) or the carcass (Table 2). Lean Rats
In the lean rats, the high-protein diets also caused a lower reduction in food intake, than that observed in the obese rats (Table 2). The total energy retention of the lean rats was always less than that of the obese rats. It is known that the efficiency of energy retention in the lean rats is much less than that of the obese rats.‘.2 Our results show that when the level of dietary proteins increased, energy retention decreased, and that the decrease was greater in the lean than in the obese rats. In the lean rats on a high-protein diet, as in the obese rats, liver phosphoenolpyruvate carboxykinase activity increased, hepatic glycogen content decreased, and blood glucose remained at a normal level. Nitrogen retention decreased more in lean rats than in obese rats. Therefore, we do not agree with Deb et al’ that the Fa/rats used dietary protein more efficiently than the obese rats. Furthermore, with a protein intake 50% lower than in obese rats (Fig. 4), hepatic urea concentration was similar in both groups. In the Fa/ - rats, high-protein diets did not produce a decrease in plasma nonesterified fatty acid concentration, glycerol, and triacylglycerols, or a rise in blood cholesterol as in the obese rats (Fig. 1). The lipid gain was reduced by half with the HP-LC diet and to zero with the HP-CF diet. When the diet provided some carbohydrates, the lean rats were able to maintain fat
206
PERET ET AL
synthesis, but when carbohydrates were totally absent from the diet, the body was unable to maintain lipogenesis in the liver. That is in agreement with hepatic acetyl-CoA carboxylase activity which was markedly decreased with the high-protein diets, more than in obese rats. The HP-CF diet produced an approximately one-third decrease in the lipid content of the carcass and no weight gain. This is in contrast to what happens in Wistar rats,14 which also show an arrest in weight gain when they are changed from a standard
diet to a carbohydrate-free diet. With time, however, the Wistar rats adapt; their food intake goes up and weight gain resumes, although weight gain lost during the first few days of the new diet is not made up. It seems, therefore, that the metabolism of lean Zucker rats cannot-in contrast with the metabolism of Wistar rats and obese Zucker rats-adapt to a high protein diet. It will be of interest to ascertain the possible metabolic disorder which these lean rats may have.
REFERENCES 1. Bray GA, York DA, Swerlolf RS: Genetic obesity in rats. I. The effects of food restriction on body composition and hypothalamic function. Metabolism 22:435442, 1973 2. Pullar JD, Webster AJF: Heat loss and energy retention during growth in congenitally obese and lean rats. Br J Nutr 31:377-392, 1974 3. Zucker LM: Efficiency of energy utilization by Zucker hereditarily obese fatty rat. Proc Sot Exp Biol Med 148498500, 1975 4. Deb S, Martin RJ, Hershberger: Maintenance requirement and energetic efficiency of lean and obese Zucker rats. J Nutr 106:191-197,1976 5. Bray GA, York DA: Studies on food intake of genetically obese rats. Am J Physiol 223: 176-l 79, 1972 6. Dilettuso B, Wangsness P: Effect of age on hyperphagia of genetically obese Zucker rats. Proc Sot Exp Biol Med 154:1-S, 1977 7. Cleary MP, Vasselli JR, Greenwood MRC: Development of obesity in Zucker obese (fafa) rat in absence of hyperphagia. Am J Physiol238:E284-E292, 1980 8. Deb S, Martin R: Effects of exercise and of food restriction on the development of spontaneous obesity in rats. J Nutr 105:543-549, 1975 9. Triscari J, Bryce GF, Sullivan AC: Metabolic consequences of fasting in old lean and obese Zucker rats. Metabolism 29:377-385, 1980 10. Bray GA: The Zucker fatty rat: A review. Fed Proc 36:148153,1977 11. Apfelbaum M, Boudon P, Lacatis D, et al: Effets mitaboliques de la diete protidique chez 41 sujets obbses. Press M&d 78:1917-1920,197O 12. Mac Kay EM, Barnes RH, Carne HO: The influence of a diet with a high protein content upon the appetite and deposition of fat. Am J Physiol 135:193-201, 1941 13. Peret J, Chanez M, Cota J, et al: Effects of quantity and quality of dietary protein and variation in certain enzyme activities on glucose metabolism in the rat. J Nutr 105:1525-1534, 1975 14. Peret J, Cota J, Chanez M, et al: Les effets de la quantite et de la qualite des proteines du regime sur la croissance, la composition corporelle et la variation des activitts de certaines enzymes du metabolisme du glucose chez le rat en croissance. Ann Biol Anim Biochim Biophys 18:663-679, 1978 15. Felig P: Four questions about protein diets. N Engl J Med 298:1025-1026,1978 16. Scheen AJ, Luyckx AS, Scheen-Lavigne MC, et al: Hormonal and metabolic adaptation to protein-supplemented fasting in obese subjects. Int J Obesity 6:165-174, 1982 17. Zucker LM, Zucker TF: Fatty, a new mutation in the rat. J Heredity 52:275-278, 1961 18. Bernhardt FW, Tomarelli RM: A salt mixture supplying the
national research council estimates the rat. J Nutr 89:495-500, 1966
of the mineral
requirements
of
19. Peret J, Macaire I, Chanez M: Schedule of protein ingestion, nitrogen and energy utilization and circadian rhythm of hepatic glycogen, plasma corticosterone and insulin in rats. J Nutr 103:866874,1973 20. Werner W, Rey HG, Weilinger M: Properties of new chromogen for the determination of glucose in blood according to the GOD/POD (glucose oxydase peroxydase). Methods Z Anal Chem 252:224-228, 1970 21. Soloni FG, Sardina LC: Calorimetric microdetermination free fatty acids. Clin Chem 19:419-424, 1973
of
22. Eggtein M, Kreutz FH: Eine neue Bestimmung der Neuralfette in Blutserum and Bewbe. Klin Wschr 44:262, 1966 23. Neri BP, Frings CS: Improved method for determination triglycerides in serum. Clin Chem 19:1201-1202, 1973 24. Wachter Lipoidphosphors
H: Ein methodischer Beitrag zur Bestimmung in Serum. Arztl Lab 11: 1 l-l 5, 1965
25. Ferro PV, Ham AB: Rapid determination cholesterol in serum. Am J Clin Path 33:545-549, 26. Bergmeyer HU: Methods York, Academic Press, 1974.
in enzymatic
of des
of total and free 1960
analysis
(ed 2). New
27. Lowry, OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 28. Hsu RY, Lardy HA: Malic enzyme, in Lowenstein JM (ed): Methods in Enzymology, New York, Academic Press 17:230-235, 1969 29. Chang HC, phosphoenolpyruvate.
Lane MD: The enzymatic J Biol Chem 241:2413-2420,
30. Maeda H, Ineda I, Sugano esis enzymes in rats fed threonine 12:61-67,1975
carboxylation 1966
of
M: Behaviour of liver key lipogenrebalanced diet. Nutr Rep Intern
31. Abraham J, Morin-Jomain M. Peretianu J: Nouvelle technique de determination de la composition corporelle des animaux de laboratoire. Bull Sot Chim Biol46:755-758, 1964 32. Snedecor GW, Cochran WG: Statistical State University Press Ames, 1974
methods.
33. Bell GE, Stern JS: Evaluation of body composition obese and lean Zucker rats. Growth 41:63-80, 1977
Iowa, Iowa of young
34. Bach A, Bauer M, Schirardin H: Data on lipid metabolism the genetically obese Zucker rat. Life Sci 20:541-550, 1977
in
35. Peret J, Foustock S, Chanez M, et al: Hepatic metabolites and amino acid levels during adaptation of rats to a high protein, carbohydrate-free diet. J Nutr 111:1704-1710, 1981 36. Martin RJ: In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats. Life Sci 14:1447-1453,1974
HIGH-PROTEIN
DIETS
IN ZUCKER
RATS
37. York DA, Bray GA: Adipose tissue metabolism in six week old fatty rats. Horm Metab Res 5355-360, 1973
38. Munro HN: General aspects of the regulation of protein metabolism by diet and by hormones, in Munro HN, Allison JB (eds): Mammalian Protein Metabolism. New York, Academic Press, 1964, pp. 38448 1 39. Radcliffe JD, Webster AJF: Regulation of food intake during growth in fatty and lean female Zucker rats given diets of different protein content. Br J Nutr 36:469, 1976 40. Jenkins TC, Hershberger TV: Effect of diet, body type and sex on voluntary intake, energy balance and body composition of Zucker rats, J Nutr 108:124-136, 1978 41. Bray GA, York DA: Hypothalamic and genetic obesity in
207
experimental animals: an autonomic and endocrine hypothesis. Phys Rev 59:719-809, 1979 42. Radcliffe JD, Webster AJF: Sex, body composition and regulation of food intake during growth in the Zucker rat. Br J Nutr 39:483-492, 1978 43. Dunn, MA, Hartsook EW: Comparative amino acid and protein metabolism in obese and non-obese Zucker rats. J Nutr 1101186%1879,198O 44. Briggs S, Freedland RA: Effect of dietary protein level on urea synthesis in isolated rat hepatocytes. J Nutr 107:561-566, 1977 45. Robinson JL, Foustock S, Chanez M, et al: Circadian variation of liver metabolites and amino acids in rats adapted to a high protein, carbohydrate-free diet. J Nutr 1I I:171 l-1720, 1981
Effects
of High-Protein
Diets in Zucker Rats
Jean Peret, And& C. Bach, Brigitte Delhomme, Brigitte Bois-Joyeux, Marc Chanez, and Henri Schirardin The effects of dietary protein on the metabolism of proteins, carbohydrates, and especially, lipids were investigated in genetically obese Zucker rats and their lean siblings. For 46 days the rats received diets containing 15%. 64%. or 82% protein, included at the expense of cornstarch. In the obese animals, the high-protein diets led to decreased food intake and weight gain. While these diets decreased the activities of lipogenic enzymes along with the lipid gain, they did not decrease the final body-fat content. The increased protein intake stimulated hepatic ureogenesis and gluconeogenesis. Lipolysis was stimulated, as demonstrated by an accumulation of ketone bodies in the liver. Blood levels of triacylglycerols, free glycerol, and nonesterified fatty acids were concomitantly decreased, which suggests an accelerated turnover of lipids. Whatever the composition of the diet, total energy retention of the lean rats was always less than that of the obese rats. The changes observed on high-protein diets were essentially the same for the two groups, except that the final body-content of lipids in the lean rats was significantly lower. In the absence of exogenous carbohydrate, the lean rats were barely able to retain nitrogen and to maintain hepatic lipogenesis. Unlike the rats from other strains, the lean Zucker rats could not adapt to a low-carbohydrate diet: this failure may be due to a metabolic disorder.
EVERAL
STUDIES
have shown that the effi-
S ciency of energy storage is higher in obese Zucker
rats (fa/fa) than in their lean littermates (Fa/-),lm4 and that the hyperphagia of fa/fa rats5’6 is a major factor determining the deposition of excess lipid. However, neither pair-feeding of obese fatty rats to the food intake of lean anima1s,‘-337 or restriction of food intake,’ or prolonged fasting’ leads to a normalization of body-fat content or elimination of other defects in these animals.” For the past decade,” the use of high-protein diets has been advocated for the treatment of human obesity. Replacing carbohydrates with proteins in the diet of Wistar rats decreases their weight gain, body lipids, and hepatic 1ipogenesis.‘2-‘4 However, not all authors regard the use of low-carbohydrate, high-protein diet as desirab1e.‘5,‘6 Therefore, this study on the effect of high dietary protein on the deposition of fat in the genetically obese Zucker rat was initiated.” Two highprotein diets were examined; one provided some carbohydrates and the other was devoid of carbohydrates. The results obtained showed that the high-protein diets lead to decreased food intake and weight gain, but they did not decrease the final body-fat content. MATERIALS AND METHODS
Corporation (Cleveland, OH, USA). Cornstarch was obtained from Roth (Karlsruhe, West Germany) and sunflower oil from the Societe des Produits Bertrand (Crigny, France). Sodium pentobarbital (Nembutal) was from Abbott (Saint-R&my-sur-Avre, France). Plasma-free glycerol and triacylglycerol, phospholipids, and total cholesterol were determined with analysis kits as follows: triglycerides, Merckotest (E. Merck, Darmstadt, West Germany); phospholipids B-test (Wako Pure Chemical Industries, Osaka, Japan); cholesterol C-test (Wako Pure Chemical Industries, Osaka, Japan). The other enzymatic reagents were from Boehringer (Mannheim, West Germany).
Animals The lean (Fa/-) and obese (fa/fa) male Zucker rats were obtained at 6 weeks of age from CSEAL-CNRS (Orleans-la-Source, France). They were kept in individual Makrolon cages in a room at 22°C + 2OC, with a 12/ 12 h light/dark cycle (lights on at 7 AM).
Experimental
Design
The rats were fed a standard UP-HC diet (Table 1). After a period of adaptation of eight days they were distributed into three groups of obese and three groups of lean rats. Each group consisting of eight rats received one of the diets described in Table I. We refer to the diets as UP-HC (usual-protein, high-carbohydrate), HP-LC (high-protein, low-carbohydrate) and HP-CF (high-protein, carbohydrate-free). Food and tap water were provided ad libitum for 40 days. During this period the weight and daily food intakes of each rat were recorded.
Materials
Sample Collection
Salt mixture,” vitamin mixture,” cellulose non-nutritive bulk, and casein were purchased from the United States Biochemical
At the end of the experimental period and during three days, one obese and one lean animal from each group were anesthetized with sodium pentobarbital (6 mg per 100 g of body weight) and killed, between 9 AM and lo:30 AM. A small sample of liver was taken by freeze-clamping. Blood was taken from the inferior vena cava in a heparinized syringe. Then other samples of liver were taken and frozen. The carcass, without blood and liver, was weighed and frozen for analysis of the final body composition. Before the start of the experiments the initial body compositions of eight young obese and eight young lean rats were determined.
From the Centre de Recherches sur la Nutrition du CNRS. Meudon-Bellevue, France, and the Laboratoire de Pathologie Generate de la Clinique Medicate A, Hbpital Civil. Strasbourg, France. Supported in part by a grant from the Instiiut National de la Sante et de la Recherche Medicate (grant N” 81.7002). Address reprint requests to Dr. Jean Peret. Cenire de Recherches sur la Nutrition du CNRS, 9 rue Jules Hetzel, 92190 MeudonBellwue, France. 0 1984 by Grune & Stratton, Inc. 0026-0495/84/3303-0002$01.00/0
200
Analytical
Methods
Glucose,*o nonesterified fatty acids?’ glycerol,2’ triacylglycerols,” phospholipids.24 and total cholesterolz5 were estimated in the plasma.
Metabolism, Vol 33, No 3
(March),
1984
HIGH-PROTEIN
DIETS IN ZUCKER RATS
201
Table 1, Composition
of Diets
UP-HC
Tukey-Hartley-Keuls
HP-LC
Diet
HP-CF
g/loo!3
RESULTS
Casein
16.3
69.4
88.8
Cornstarch
72.5
19.4
0
Sunflower oil
3.5
3.5
3.5
Salt mixture
4.3
4.3
4.3
Vitamin mixture
1.7
1.7
1.7
Cellulose non-nutritive bulk
7.7
1.7
1.7
Energy Intake; Body Weight Gain
With increased protein content in the diet obese rats reduced their food intakes (HP-LC -24%, HPCF -35%, compared with UP-HC) (Table 2). Despite the decrease in food ingested, the daily protein intake increased considerably. This results in a reduced rate of weight gain relative to the UP-HC rats (-29% and - 53%, respectively). On all the diets the lean rats ate less and gained less weight than the obese rats. Compared to lean rats fed the usual diet, those on the high-protein diets had reduced food intake (-40% and -45%, respectively), increased protein intake, and reduced daily weight gain.
kJ/lOO g diet Energy value
1596.2
1525.9
1499.1
% metabolizable energy Protein
15.7
70.1
91.2
Carbohydrate
76.0
21.3
0
8.3
8.6
Fat
8.8
UP, usual protein; HP, high protein: HC, high carbohydrate; LC, low carbohydrate: CF. carbohydrate-free.
Casein was “vitamin-free”
as it
was 92% protein. The diets contained 15% (UP-HC), 64% (HP-LC). and 82%
(HP-CF) protein. Energy values of protein, starch, and oil were
assumed to be 16.74, @-hydroxybutyrate, tamine,
urea,
16.74,
and 37.66
acetoacetate,
inorganic
kJ respectively.
lactate,
phosphate,
pyruvate,
ATP,
ADP,
Body Composition;
and AMP
Water
(by lyophilisation),
portions of liver.
methanol
(2/l,
v/v),
total cholesterol”
protein,”
Lipids
were
Whereas in the obese rats the amount of protein in the food ingested increased from 15% to 82%, the energy retention decreased by 43%, the nitrogen retention decreased by 78%, and the daily lipid gain decreased by 39% (Table 2). In terms of final body composition, the effect of the dietary protein content was so slight that none of the changes in the percentages of water, protein, or lipid were significant. As decribed previously,2,33 the carcasses of the lean rats contained more water, more protein, and less lipid per 100 g than those of the obese rats (Table 2). In the lean rats, the I-II’-CF diet led to a decrease in body lipid content (-33% compared with HP-HC). The
and lipid were estimated on
were extracted
and triacylglycerols.23
enolpyruvate
with chloroform/
phospholipids,24 and
were determined.
The hepatic activities of mafic enzyme (EC carboxykinase
(EC
4.1 .1.32),*9
I .l. l.40)?’
phospho-
and acetyl-CoA
car-
boxylase, (EC 6.4. I .2)” were measured at 37°C. The activities are expressed per milligram of cytosol protein, or per total liver. The content of water, total lipid, and protein were determined the rat carcasses according to the method of Abraham energy retention,
nitrogen
retention,
in
et al.” The
and lipid gain were derived
from the differences between the initial and final body compositions and were expressed per day of the experiment. Statistical
analysis of results (SEM,
Table 2.
analysis of variance,
Effect of Dietary Protein on Weight
and
Gain, Energy Intake, Final Body Composition. Nitrogen,
lean
224.5
Weight gain (g/d) Final body Composition (g/ 100 9) Energy retention (kJ/d) Nitrogen retention (mg/d) Lipid gain (g/day)
* 6.8”,**
2.6 + O.l”** (
Water protein (N x 6.25) llpid
67.7
* 0.5*’
19.2 +_0.5-9 7.0 + 0.3*,** 20.3
r 1.2’”
75 + 6’ 0.22
f 0.02”‘f’
Retention
of Body Energy and
end Lipid Gain
UP-HC
Energy intake (kJ/d)
Energy and Nitrogen Retention;
Lipid Gain
gfycogen, glu-
measured’” in liver samples taken by freeze-clamping. other
test) was carried out according to Snedecor
and Cochran.”
HP4C obese
354.8
t 5.6”
4.5 t 0.3” 38.7
f 0.8
15.8 1 0.5
lean
134.4
t 5.gb.**
0.7 r o.2b,” 69.2
* 0.8.’
18.0 c 0.7’
HP-CF obese
269.0
_t 9.2”
3.2 + 0.2” 37.7
lean
123.5
obese
* 5.7b.‘r
0.1 + 0.2’,.
232.1 l
-t 11.7”
2.1 + 0.2’
t 1.1
66.9
A 0.9**
37.9
15.8 i 0.4
22.4
A 1.1”
14.9 * 0.4
+ 1.0
41.5
r 1.3
6.8 + 0.5’**
44.7
? 1.7
4.7 i 0.5b.‘f
42.6
+ 1.4
100.1
f 5.8”
6.9 ? 1.6b.*’
85.7
2 6.2”
1.1 * 2.0c,**
56.5
t 2.8’
93 + 9’ 2.31
+ 0.15^
23 * qb+’ 0.11
+ 0.03b.*”
62 + 8” 2.03
* 0.15”
1 + 6’,** + 0.03’~“’
0.02
20 + 3’ 1.44 + 0.08Y
Results are expressed as means & SEM for (8) rats fed each diet for 40 days. Initial body weight, body energy, body nitrogen, and body lipid averaged 195 + 6 g, 3262
r 142 kJ, 5.7 + 0.1 g, and 64.2 + 3.2 g. respectively for obese rats and 142 + 4 g, 967 * 43 kJ, 4.6 ? 0.2 g, and 7.4 + 0.5 gfor lean rats. UP-HC. 15% protein; HP-LC. 64% protein; HP-CF, 82% protein. Statistical significance: between lean and obese, “P < 0.05, l*P < 0.01 (analysis of variance): between diets, means not followed by the same superscript letter are significantly different (Test Tukey-Hartley-Keuls) (P < 0.05, a, b, and c (in order of decreasing magnitude) in the case of lean rats and x, y, and z in the case of obese rats.
202
PERET ET AL
daily lipid gain of lean rats was not only less than that of the obese rats, but it was also reduced by half with the HP-LC diet, as compared with the UP-HC diet, and was reduced to zero with the HP-CF diet. The total energy retention and nitrogen retention were also lower in lean rats than in obese rats, and they were zero with the HP-CF diet. Close examination of the data with the HP-CF diet shows that some lean rats lost weight and at the end of the experiments had slightly negative values for energy retention, nitrogen retention, and lipid gain, while others gained a little weight and accumulated some nitrogen and fat. These differences would be explained by a difference in the genotype. The lean rats were not identified as Fa/Fa homozygotes or Fa/fa heterozygotes; the effect of a high-protein, low-carbohydrate diet may differ slightly between these two types. Plasma Glucose and Lipids Plasma glucose was similar in lean and obese rats on each diet (Fig. 1).
In obese rats, HP-LC and HP-CF diets produced a decrease in plasma triacylglycerol ( -43% and - 40%, respectively), free glycerol (- 21% and - 3.5%,), and nonesterified fatty acids (-24% and -45%), and a slight increase in blood cholesterol (+ 10% and + 24%) when compared with the UP-HC diet. The levels of triacylglycerol, free glycerol, cholesterol, and phosphohpids were significantly lower in the lean rats than in the obese rats.34 The values in lean rats were marginally affected by the composition of the diet. Liver Weight
and Liver Composition
The hepatomegaly of the obese rats was not reduced by high-protein diets (Fig. 2). The weight of liver per 100 g of body weight, although higher in obese than in lean rats, did not vary with the composition of the diet (data not shown), both in fa/fa or in Fa/rats. Therefore, a gram of liver can be considered as a metabolic unit, which makes simple the comparison 61l r
3
5,
i
OL
I
z
E
,
Y
250.
0
1
1
L
64
, a2
IS
PROTCIN
LEVEL
1
L
64
4 a2
~/lOOgd~et
Fig. 1. Effect of dietary protein on plasma glucose and lipids. Results are expressed as mean k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Triacylglycerol and phospholipid levels were calculated using average molecular weights of 885 and 801, respectively. Nonesterified fatty acids (NEFA) (expressed as pelmitic acid equivalents). Open squares, lean; closed squares. obese. UP-HC. 15% protein: HP-LC. 84% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
I
I
15
64 PROTEIN
a2 LEVEL
15 g/100,
I
64
a2
d,et
Fig. 2. Effect of dietary protein on liver weight, glycogen, and lipids. Results are expressed as means k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares, lean; closed squares, obese. Glycogen and lipids are reported as per gram wet liver. UP-HC. 15% protein; HP-LC. 84% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
HIGH-PROTEINDIETS IN ZUCKER RATS
203
between the hepatic parameters of the two types of rat. Lipid (Fig. 2) and protein (data not shown) compositions of the liver did not change in obese or lean rats in response to high-protein diets. The concentration of glycogen decreased less in obese rats (-26% with HP-LC, and -38% with HP-CF, in comparison with UP-HC) than the lean rats (- 38% and -48%, respectively). Hepatic Metabolites
High-protein diets did not produce any significant change in hepatic lactate or pyruvate concentrations both in obese or lean rats (Fig. 3). The decrease in lactate/pyruvate ratio in obese rats receiving high protein diets was not statistically significant. High protein diets produced an increase in hepatic concentration of ,&hydroxybutyrate (+ 194% with HPLC, +23 1% with HP-CF, in comparison with UP-HC) in obese rats without a parallel change in acetoacetate
(Fig. 3). In contrast, the change in hepatic P-hydroxybutyrate concentration was less marked in lean rats ( + 126% and + 132%, respectively) and was accompanied by an increase in the concentration of acetoacetate (+ 102% and +94%, respectively). The increase in total ketone bodies @I-hydroxybutyrate and acetoacetate) was of the same magnitude both in lean and obese rats (+9 1% and + 121% in the obese rats for HP-LC and HP-CF, respectively, compared with + 117% and + 119% in the lean rats). In contrast, the ratio of a-hydroxybutyrate to acetoacetate was affected differently by high protein diet in obese and lean rats: this ratio tripled in the obese rats receiving a high-protein diet, while it remained unchanged in the lean rats in the same nutritional conditions. In parallel with the increased protein intake, an
x
;il: x
a**
**b
Y
c 1 ! 1750_
*c
f
6_
3.
b
Y
I
15
!
64 PROTEIN
82
0
I
15
I
64
82
LEVELg/lO0~d~~~
Fig. 3. Effect of dietary protein on liver metabolites. Results are expressed as mean k SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares, lean; closed squares. obese. The metabolites are reported as per gram wet liver. UP-HC. 15% protein; HP-LC, 64% protein; HP-CF. 82% protein. @-OH& @-hydroxybutyrate; AcAc, acetoacetate. For statistical analysis see legend of Table 2.
1
64
15 PROTEIN
LEVEL
82
g/1009 d,e,
Fig. 4. Effect of dietary protein on protein intake and on liver urea and glutamine. Results are expressed as mean f SEM for eight rats fed each diet for 40 days. Bars represent 1 SEM. Open squares. lean; closed squares, obese. Urea and glutamine are reported as per gram wet liver. UP-HC. 15% protein: HP-LC. 54% protein: HP-CF. 82% protein. For statistical analysis see legend of Table 2.
204
PERET ET AL
Table 3. Effect of Dietary Protein on Liver Adenine Nucleotides, Inorganic Phosphate (Pi), and Phosphorylation State Metabolites(nmol/g) UP-W
Metabolites (nmollg) ATP
2.142
f 74
745 + 65b
AMP
165 f 18b
ATP ADPMPiM-’
HP-CF
lean
obese
ADP Pi
HP-CC
lean
obese
lean
obese
1,738
+ 100
1,924
+ 93
1,759
+ 126
2.071
f 82
1,759
r 111
1.007
f 126
1,089
k 55”
1,109
+ 63
1,171
+ 64”
1,029
+ 48
177 ) 26’
265 + 29”
1,910
f 125h
1,892
t 168’
1,626
f 180
1,108
+ 207”
2,191
343 + 459
r 173”’
3.198
662 + 80b
260 + 32’
i 291”
2.787
548 + 83”
289 + 54w
i 202’
2,815
684 + 8gb
2 126’
606 f 72”
Results are expressed as means for eight rats fed each diet for 40 days. UP-HC, 15% protein; HP-LC, 64% protein; and HP-CF. 82% protein. For statistical analysis see legend of Table 2.
accumulation of urea was observed in the liver (Fig. 4).35 It is noteworthy that the lean rats accumulated as much as the obese rats, even though lean rats ingested only half the amount of protein as obese rats. The decrease in liver glutamine content was also identical in the two phenotypes. Hepatic Adenine Nucleotides Hepatic concentrations of adenine nucleotides were the same for the obese as for the lean rats (Table 3). The high-protein diets increased the AMP and inorganic phosphate content in both groups of animals and the ADP content in the lean rats. In addition, when the dietary level of protein increased, the state of phosphorylation decreased. Activities of Malic Enzyme, Acetyl-CoA and Phosphoenolpyruvate
Carboxylase,
Carboxykinase
In the obese rats, the high-protein diets produced a decrease in the activity of acetyl-CoA carboxylase and of malic enzyme, and a rise in the activity of phosphoenolpyruvate carboxykinase (Table 4). The activities of acetyl-CoA carboxylase and malic enzyme were lower in lean than in obese rats. In the lean rats the high-protein diets increased the activity of phosphoenolpyruvate carboxykinase and strongly reduced that of acetyl-CoA carboxylase.
DISCUSSION Obese Rats
The high activities of malic enzyme and of acetylCoA carboxylase (Table 4) in the liver of obese rats receiving the UP-HC diet suggest that lipid synthesis is greatly accelerated in the liver? and, also, in the adipose tissue3’ of these animals. This results in an accumulation of triacylglycerol in the liver (Fig. 2), in the carcass, (Table 2), and in the blood (Fig. 1). When the carbohydrate content of the diet was reduced (HP-LC diet) or eliminated (HP-CF diet) and replaced with proteins, it would have been logical to expect that both lipogenesis and accumulation of fat would have been reduced since carbohydrates are good lipogenic precursors. We also observed that highprotein diets decreased the daily weight gains (Table 2). Munro3* attributed this decrease to the inability of high-protein diets to ensure an adequate energy supply * This reduction of weight gain can be explained by the decrease in energy intake following the passage from a normal-protein diet to a high-protein diet (Table 2). The HP-CF diet appeared to be particularly effective; it reduced the food intake by about a third and the daily weight gain by half. Nevertheless, these animals remained obese at the end of the experiments, although they were less fatty than those receiving the UP-HC diet (Table 2). In fact, the daily lipid gain was
Table 4. Effect of Dietary Protein on Hepatic Enzyme Activities HP-LC
UP-HC lean
Enrvmes
ACC ME PEPCK
pmolltotal liver
11.8 + 2.9’+*
30.6
-e 3.4”
21.3
f 2.3’
HP-CF
lean
obese
5.5 t l.gb,** 7.5 + 2.3b.**
lean
obese
21.0
+ 2.3”
13.1 * 0.7*
4.3 + O.gb.*+ 5.9 k l.ob.+*
obese
18.2 zk 3.6”
nmol/mg proteins
18.3 + 2.9”“*
fimolltotal liver
35.9
k 2.5b”
214.4
f 18.8”
52.1
+ 2.8”.”
153.4
* 10.6”
33.5
f 3.gb.**
151.9
12.1 + 1.9” k 8.3”
nmol/mg proteins
59.8
r 5.0**
146.2
k 10.9’
66.3
+ 7.6+*
101.6
t 7.2’
57.2
+ 3.1”
101.6
+ 5.5”
pmol/total liver
11.3 + l.lb**
17.8 k 1.2”
26.0
k 2.4”
28.2
+ 3.3”
25.1
+ 3.0”
37.0
t 6.5’
nmoljmg proteins
17.1 + 1.5b
15.7 * 0.8’
40.9
i- 3.01,+*
21.1
t 1.9’
44.3
+ 3.1a**
28.2
+ 3.9’
Results are expressed as means -c SEM for eight rats fed each diet for 40 days. ACC, acetyl CoA carboxylase; ME, malic enzyme: PEPCK, phosphcenolpyruvate carboxyidnase. UP-HC. 15% protein: HP-LC, 64% protein: HP-CF. 82% protein.
For statistical
analysis see legend of Table 2.
HIGH-PROTEIN
DIETS IN ZUCKER RATS
reduced at best by a third (with the HP-CF diet), and the activities of the two enzymes involved in hepatic lipogenesis, acetyl-CoA carboxylase and malic enzyme, were also reduced by about a third (Table 4). The final body composition was the same whatever the protein content of the diet. Radcliffe and Webster39 and Jenkins and Hershberger4’ have previously reported that a high-protein diet is unable to prevent the accumulation of fat in obese Zucker rats. One wonders whether these metabolic changes are due to altered protein and carbohydrate content of the diet or to the changes in total energy intake. When obese rats on high-carbohydrate diets are pair-fed with lean rats, they still gain excess fat, for review see Bray and York.4’ Moreover, Radcliffe and Webster,42 by comparing gains in protein, lipid, and energy in obese and lean male rats pair-fed containing 10% and 50% protein, showed that the greater rate of lipid deposition in obese rats was not due to hyperphagia per se, but to an abnormal partition of metabolic energy between fat (too much) and protein (too little). Thesedata strongly suggest that the metabolic changes observed in the present study can be related to altered protein and carbohydrate composition of the diet and to the metabolic defects of obese rats. The inability of high-protein diets to reduce the percentage of fat in the carcass lies in two observations. The first, made by Radcliffe and Webster,39 is that whatever the level of protein in the diet, the obese rats maintained highly efficient energy metabolism. This is seen in the case of the HP-CF diet, with 57 kJ of energy retention per day (Table 2). Dunn and Hartsook showed by the use of radioactive tracers that the obese Zucker rat can incorporate a larger proportion of dietary amino acids in their body lipids than can the lean rats. Lipogenesis from amino acids requires that amino acids were first deaminated. Our data show that the increase in ingested proteins in obese rats is accompanied both by an increase in liver urea (Fig. 4)44 and by a decrease in nitrogen retention (Table 2). The concomitant decrease in liver glutamine in the rats receiving the high-protein diets suggests a use of this amino acid in the gluconeogenesis pathway after it has been deaminated. An increase in gluconeogenesis is essential for an organism that receives little or no exogenous carbohydrate. The extent of this stimulation is demonstrated by the increased activity of liver phosphoenolpyruvate carboxykinase (Table 4). It appears that the rise in gluconeogenesis enables the maintenance of a normal blood glucose level (Fig. 1) without preventing the fall in hepatic glycogen concentration (Fig. 2). The rates of ureogenesis and gluconeogenesis are
205
increased by high-protein diets whereas glycolysis is decreased.45 This could explain the increase in the hepatic levels of ADP, AMP, and inorganic phosphate and hence the decreased phosphorylation ratio observed in HP-LC and HP-CF rats (Table 3).35 As previously reported for Wistar rats,35l45 highprotein diets are ketogenic in Zucker rats. In this study hepatic @hydroxybutyrate tripled (Fig. 3). The increase in ketogenesis involves an acceleration of tissue lipolysis. The fact that blood triacylglycerols, free glycerol, and nonesterified fatty acids (Fig. 1) were concomitantly decreased can be explained only by an accelerated turnover of lipids. The cholesterol response was different. In the obese rats the diets poor in carbohydrates, and also in lipids, led to an increased level of blood cholesterol. We think that a fraction of the acetyl-CoA released by lipid catabolism is prevented from being totally oxidized in the Krebs cycle because oxaloacetate is decreased due to gluconeogenesis. This acetyl-CoA may contribute both to stimulating ketogenesis, as discussed above, and to increasing the synthesis, and hence, the blood level, of cholesterol. These losses are not enough, however, to reduce the lipid content of the liver (Fig. 2) or the carcass (Table 2). Lean Rats
In the lean rats, the high-protein diets also caused a lower reduction in food intake, than that observed in the obese rats (Table 2). The total energy retention of the lean rats was always less than that of the obese rats. It is known that the efficiency of energy retention in the lean rats is much less than that of the obese rats.‘.2 Our results show that when the level of dietary proteins increased, energy retention decreased, and that the decrease was greater in the lean than in the obese rats. In the lean rats on a high-protein diet, as in the obese rats, liver phosphoenolpyruvate carboxykinase activity increased, hepatic glycogen content decreased, and blood glucose remained at a normal level. Nitrogen retention decreased more in lean rats than in obese rats. Therefore, we do not agree with Deb et al’ that the Fa/rats used dietary protein more efficiently than the obese rats. Furthermore, with a protein intake 50% lower than in obese rats (Fig. 4), hepatic urea concentration was similar in both groups. In the Fa/ - rats, high-protein diets did not produce a decrease in plasma nonesterified fatty acid concentration, glycerol, and triacylglycerols, or a rise in blood cholesterol as in the obese rats (Fig. 1). The lipid gain was reduced by half with the HP-LC diet and to zero with the HP-CF diet. When the diet provided some carbohydrates, the lean rats were able to maintain fat
206
PERET ET AL
synthesis, but when carbohydrates were totally absent from the diet, the body was unable to maintain lipogenesis in the liver. That is in agreement with hepatic acetyl-CoA carboxylase activity which was markedly decreased with the high-protein diets, more than in obese rats. The HP-CF diet produced an approximately one-third decrease in the lipid content of the carcass and no weight gain. This is in contrast to what happens in Wistar rats,14 which also show an arrest in weight gain when they are changed from a standard
diet to a carbohydrate-free diet. With time, however, the Wistar rats adapt; their food intake goes up and weight gain resumes, although weight gain lost during the first few days of the new diet is not made up. It seems, therefore, that the metabolism of lean Zucker rats cannot-in contrast with the metabolism of Wistar rats and obese Zucker rats-adapt to a high protein diet. It will be of interest to ascertain the possible metabolic disorder which these lean rats may have.
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