Nutrient Physiology, Metabolism,

and Nutrient-Nutrient Interactions

A High-Protein, High-Fat, Carbohydrate-Free Diet Reduces Energy Intake,

Hepatic Lipogenesis, and Adiposity in Rats
Lisa Pichon, Jean-François Huneau, Gilles Fromentin,1 and Daniel Tomé
UMR INRA 914 Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique
Paris-Grignon, F75231 PARIS Cedex 05, France

ABSTRACT The aim of this work was to determine the effects in rats of ingesting 1 of 3 diets with normal or high protein
concentrations and various carbohydrate:lipid ratios on weight gain, body composition, and the development and
metabolism of white adipose tissue (WAT). For this purpose, male Wistar rats were fed for 20 or 42 d a high-
carbohydrate, low-fat, normal-protein diet (76, 10, and 14% of energy as carbohydrate, lipid, and protein, respectively,
carbohydrate:lipid ratio (C/L) ¼ 7.6), a normal-carbohydrate, low-fat, high-protein diet (35, 10, and 55% of energy as
carbohydrate, lipid, and protein respectively, C:L ¼ 3.5), or a carbohydrate-free, high-fat, high-protein diet (45 and 55%
of energy as fat and protein, respectively, C:L ¼ 0). Growth, food intake, body composition, WAT cellularity, and several
markers of lipogenesis including fatty acid synthase and lipoprotein lipase activities were measured in adipose tissue
and liver. Lowering the C:L ratio reduced the development of WAT, weight gain, body fat mass, and adipocyte size, and
in rats fed the carbohydrate-free diet (C:L ¼ 0), the total number of adipocytes in subcutaneous WAT. These reductions
in adipose tissue development with decreases in the C:L ratio of the diet seemed to be due primarily to reduced hepatic
lipogenesis. J. Nutr. 136: 1256–1260, 2006.

KEY WORDS:
adipocytes
body composition
fatty acid synthase
lipoprotein lipase

carbohydrate-free diet

The precise role of different macronutrient combinations,
i.e., the relative effects of carbohydrate, fat, and protein on total
energy intake, energy metabolism, and adiposity, remains a sub-
ject of debate. Increasing the protein content in the diet usually
reduces energy intake and fat deposition (1–5). Different studies
also showed the importance of the carbohydrate to fat ratio
rather than the fat content per se in the diet to the devel-
opment of adiposity (6). In this context, a high-fat diet would
lead to overfeeding and obesity when the diet is also rich in
carbohydrate, which favors insulin secretion, lipogenesis, and
fat deposition, whereas in the absence of carbohydrate, a lower
insulin response to feeding would favor lipid oxidation rather
than deposition. The present study addressed the effects of
lowering the carbohydrate:lipid (C:L)2 ratio of the diet in a con-
text of a normal or high-protein diet on energy intake, lipo-
genesis, and adiposity. For this purpose, energy intake, body
weight gain, body composition, adipose tissue cellularity, lipo-
genesis in the liver [fatty acid synthase (FAS)] and lipolysis in
the adipose tissue [lipoprotein lipase (LPL)] were determined
in rats fed one of the following: 1) a control (high-carbohydrate,
1
2
To whom correspondence should be addressed. E-mail: fromenti@inapg.fr.
Abbreviations used: AI, adiposity index; BSA, bovine serum albumin; C:L,
carbohydrate:lipid; FAS, fatty acid synthase; LPL, lipoprotein lipase; P14C76L10,
diet containing 14% protein, 76% carbohydrate, and 10% fat; P55C35L10, diet
containing 55% protein, 35% carbohydrate, and 10% fat; P55L45, diet containing
45% as fat and 55% as protein; SBAT, scapular brown adipose tissue; WAT, white
adipose tissue.
low-fat, normal-protein, i.e., a high C:L) diet, 2) a normal-
carbohydrate, low-fat, high-protein, i.e., a normal C:L ratio diet
obtained by increasing protein at the expense of carbohydrate
without any change in the fat content, or 3) a carbohydrate-
free, high-fat, high-protein, i.e., a 0 C:L ratio] diet obtained
from situation 2 by increasing the level of fat at the expense of
carbohydrate.
MATERIALS AND METHODS
Animals and diets. Male Wistar rats were housed individually in
stainless steel wire cages in a room with a 12-h light-dark cycle (lights
on: 1030–2230) and maintained at a temperature of 21 6 18C. The
rats were adapted for 10 d to a control diet providing an adequate
intake of protein (14% total milk protein) and then switched to 1 of
the 3 experimental diet (Table 1): the high-carbohydrate, low-fat,
normal-protein group (P14C76L10) was fed a diet with 76% of energy
as carbohydrate, 10% as fat, and 14% as protein. The normal-carbo-
hydrate, low-fat, high-protein group (P55C35L10) was fed a diet
providing 35% of energy as carbohydrate, 10% as fat, and 55% as pro-
tein, and the carbohydrate-free, high-fat, high-protein group (P55L45)
was fed a diet devoid of carbohydrate, providing 45% of energy as fat
and 55% as protein. Rats had free access to food and water, with fresh
food provided daily. They consumed the experimental diets for 42 d in
Expt. 1 (preliminary experiment) and for 20 d in Expt. 2.
In a preliminary experiment (Expt. 1), 24 Wistar rats, initially
weighing 310 6 5 g were acclimated and then divided into 3 groups as

0022-3166/06 $8.00 Ó 2006 American Society for Nutrition.

Manuscript received 14 November 2005. Initial review completed 23 November 2005. Revision accepted 6 January 2006.

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 CARBOHYDRATE-FREE DIET AND ADIPOSITY 1257

TABLE 1 dissolved in 10 mL glycerol], 4 volumes of 0.2 mol/L Tris HCl buffer

pH 8.1 containing 150 mmol/L NaCl and 3% BSA, and 1 volume of
Composition of the diets heated rat serum as a source of apolipoprotein CII, the natural acti-
vator of LPL. The reaction was started by mixing 100 mL of substrate
Diet P14C76L10 P55C35L10 P55L45 and 100 mL enzyme solution; it was allowed to proceed for 1 h at 378C
in a shaking water bath. The reaction was stopped by successively
Ingredient g/kg diet adding 3.5 mL of methanol:chloroform:heptane (141:125:100, by vol)
Milk protein1 140 530 720 and 1.05 mL potassium tetraborate:potassium carbonate buffer 0.1
Cornstarch2 622.4 287 0 mol/L, pH 10.5. After agitation and centrifugation (3000 3 g, 48C, 10
Sucrose3 100.3 45.7 0 min), 1 mL of the methanol alkaline phase containing the free fatty
Soybean oil4 40 40 182.7 acids was transferred to a scintillation flask and counted using liquid
AIN 93M Mineral mix5 35 35 35 scintillation. Results were expressed as nanomoles of free fatty acids
AIN 93M Vitamin mix5 10 10 10 released per 100,000 cells per hour.
a-Cellulose6 50 50 50 Liver fatty acid synthase (FAS) activity. Liver FAS activity was
Choline5 2.3 2.3 2.3 assessed using the spectroscopic method described by Halestrap and
Metabolizable energy, kJ/g 14.88 15.02 19.84 Denton (12). The liver (500 mg) was homogenized at 48C in 5 volumes
of Tris HCl buffer, pH 7.4, containing 0.25 mol/L sucrose, 1 mmol/L
1 Total Milk Protein, Armor Proteins. dithiothreitol, and 1 mmol/L EDTA. The homogenate was centrifuged
2 Cerestar. at 48,000 3 g and 48C for 2 h and the supernatant used as an enzyme
3 Eurosucre.
4
source. The reaction took place at 378C in 0.1 mol/L potassium
Bailly SA. phosphate buffer, pH 6.5, with 0.1 mmol/L NADPH,H1, 25 mmol/L
5 ICN Biochemicals.
6 acetyl CoA, and 60 mmol/L malonyl CoA. The oxidation of NADPH,
Medias filtrants.
H1 was monitored continuously by absorbance measurement at
340 nm. Proteins were assayed using the bicinchoninic acid method
(13).
described above. Growth and food intake were measured on a daily Body composition. Carcass analysis was conducted on the rats
basis for 42 d. In a second experiment (Expt. 2), 24 Wistar rats initially remaining at the end of the 20-d feeding period. The rats were
weighing 180 6 5 g were acclimated and then divided into 3 groups as deprived of food overnight and killed with an i.p. injection of 13.6
described above. On d 20, 8 rats from each group were killed for the mg/100 g body weight sodium pentobarbital (Sanofi santé animale).
determination of body composition, white adipose tissue (WAT) Blood samples were collected rapidly from the vena cava in heparinized
cellularity, and LPL and FAS activity. Blood samples were collected syringes, to prevent clotting. The abdomen was then opened and the
and plasma was stored at 2808C until analysis. Samples of retroper- liver, the two main abdominal fat pads (epididymal and retroperito-
itoneal and subcutaneous WAT (4 g) were processed immediately to neal), the subcutaneous fat pad, the interscapular BAT, and the strip-
determine adipocyte cell size distribution and fat pad cellularity, and to ped carcass were quickly removed and weighed.
measure triglyceride levels, as described below. All experiments were Statistical analysis. All results are expressed as means 6 SEM.
carried out in accordance with the recommendations of the French Body weight and food intake data were analyzed using the mixed
Committee for Animal Care. procedure for repeated measurements in the SAS software package
Adipocyte isolation and WAT cellularity assessment. Adipocyte (SAS version 8.02), with group and time as fixed effects. Generalized
isolation from retroperitoneal and subcutaneous white adipose fat pad linear model analysis was used for the comparison of body composition,
was performed by collagenase digestion, according to the Rodbell FAS and LPL activity, and the size distribution of adipocytes was
method modified by Martinson (7). Briefly, 3 g of tissue were cut with a analyzed using the x2 test.
pair of scissors and added in 10 volumes of oxygenated (O2:CO2, 95:5)
Krebs-Ringer buffer [120 mmol/L NaCl, 4.7 mmol/L KCl, 1.25 mmol/L
CaCl2, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO47H2O, 32.3 mmol/L
NaHCO3, 4% bovine serum albumin (BSA), 5 mmol/L glucose, pH
7.4], and 10 mg collagenase was added per gram of WAT. Digestion RESULTS
was allowed to proceed at 378C under mild agitation for 45 min in
polypropylene tubes. Floating adipocytes were filtered through a 190- Food intake and body weight gain. In Expt. 1, growth and
mm Nylon gauze, collected, rinsed in collagenase-free buffer, and food intake were measured daily for 42 d in rats fed the
diluted in 2.5 mmol/L Ca21Krebs-Ringer buffer. The cell suspension P14C76L10, P55C35L10, or P55L45 diet. During the adapta-
(100 mL) was gently spotted onto a microscope slide and cell diameters tion period (until d 0), rats (n 5 24) consumed 409 6 12 kJ/d
were measured on 100 adipocytes using a micrometric scale (X40). and their mean body weight was 346 6 5 g at the time of the
Mean adipocyte volume was calculated according to the method introduction of the experimental diet. During the 42-d feeding
described by Goldrick (8): V 5 p/6 3 d (d2 1 3s2), where d is the period, rats fed the P14C76L10 diet consumed more energy
mean diameter and s2 is the variance of the diameter on 100 than those fed the P55C35L10 or the P55L45 diets (group
adipocytes. The amount of lipid per milligram fat pad was measured on effect P , 0.05, time 3 group effect P , 0.001) (Fig. 1B.
a 1-g sample, using the method of Folch (9). Assuming a density of
0.915 for adipocytes, corresponding to that of a triolein droplet, the Overall, lowering the C:L ratio of the high-protein diet from 3.5
number of adipocytes per milligram of fat pad could be calculated, to 0 (P55C35L10 and P55L45 groups) did not affect energy
when the mean lipid content of one adipocyte was known. intake (P 5 0.58; P55C35L10 and P55L45), although a slight
Adipose tissue lipoprotein lipase (LPL) activity. Lipoprotein but significant reduction of energy intake occurred during the
lipase activity in retroperitoneal WAT was assessed using the method first 2 wk, followed by a gain during the subsequent period (time
described by Nilsson-Ehle and Schotz (10); LPL was extracted as 3 group effect, P , 0.01). Compared with the P14C76L10
described by Dugail (11). Retroperitoneal tissue was crushed at 48C in group, weight gain was lower in both the P55C35L10 and the
50 mmol/L NH4Cl buffer, pH 8, containing 4% BSA and 4 kIU/L P55L45 groups throughout the experimental period (group ef-
heparin. Proteins were precipitated with acetone, pelleted by centri- fect P , 0.05, group 3 time effect P , 0.01) (Fig. 1A).
fugation (3500 3 g, 20 min, 48C), rinsed 3 times with acetone, and Growth was further decreased when the G:L ratio of the diet
dried with diethyl ether. Proteins were resuspended in 50 mmol/L
NH4Cl, pH 8.1, containing 4 kIU/L heparin. Insoluble particles were was lowered from 3.5 to 0 (group effect P , 0.05). The
removed by centrifugation (3500 3 g, 20 min, 48C) and the super- difference between the P55C35L10 and the P55L45 fed rats
natant was used as a source of LPL. The substrate was prepared by was significant after only 2 d of experimental diet and remained
mixing 1 volume of solution A [600 mg triolein trace-labeled with almost constant throughout the experiment (no group 3 time
150 mCi [3H]-triolein (2 TBq/mmol, NEN) and 36 mg egg lecithin effect). In the next experiment (Expt. 2) which considered body

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 1258 PICHON ET AL.

weight by the weight of the stripped carcass) was compared, the

FIGURE 1 Cumulative body weight (A) and energy intake (B) of rats
consuming the P14C76L10, P55C35L10, or P55L45 diet ad libitum.
Values are means 6 SD, n ¼ 8. Means without a common letter differ,
P , 0.05.
composition, adipocyte cellularity, and enzymatic activities, the
initial weight was lower. The rats were also fed the experimen-
tal diets for a shorter period (20 d), but body weight gain and
food intake were similar (data not shown).
Body composition. Organ weight proportions at the end of
the experiment differed as a function of diet (Table 2). Compared
with the P14C76L10 group, all of the fat pads and the total
WAT of the P55C35L10 group were not different. But when
the adiposity index (AI, calculated by dividing total WAT
reduction in the C:L ratio from 7.6 to 3.5 reduced adiposity
because the AI was lower in the P55C35L10 than in the
P14C76L10 group (P , 0.0005). Compared with the P14C76L10
and P55C35L10 groups, P55L45 rats had lower total body fat
mass. This was also the case for each of the fat pads (epididymal,
retroperitoneal, and subcutaneous), and the AI (P , 0.0075).
Thus, reduction of the C:L ratio from 3.5 to 0 also reduced body
fat mass. These effects of the C:L ratio of the diet were also
observed for the proportion of scapular BAT. Lowering the C:L
ratio in the diet led to an enhancement of the liver proportion.
Adipose tissue cellularity. Cell size distributions in the
retroperitoneal fat pad were similar to those of the subcutane-
ous WAT (Fig. 2). Compared with the P14C76L10 group, the
adipocyte size distributions in the P55C35L10 and P55L45
groups were shifted toward smaller diameters in both retroper-
itoneal and subcutaneous tissues. This shift was more pro-
nounced in the P55L45 group than in the P55C35L10 group
because 53% of the adipocytes in the retroperitoneal fat pad
and 60% in the subcutaneous fat pad were smaller than 100
mmol/L in the former group compared with 41 and 47% in the
latter group. Consistently, the mean diameters of retroperito-
neal and subcutaneous adipocytes were significantly higher in
the P14C76L10 group than in the P55C35L10 and P55L45
groups (retroperitoneal: P , 0.0001; subcutaneous: P , 0.0006).
The number of adipocytes in retroperitoneal tissue was sig-
nificantly smaller in the P55L45 group (P , 0.0405) but did not
differ in the P55C35L10 and P14C76L10 groups (Table 3). In
subcutaneous tissue, the total number of adipocytes did not
differ among the 3 groups. The cellularity (number of adipo-
cytes/mg of tissue) of retroperitoneal and subcutaneous WAT
differed significantly among the groups, with higher values in
P55L45 rats and lower values in P14C76L10 rats.
Liver FAS and WAT LPL activities. Hepatic FAS activity
was greater in rats fed P14C76L10 than in those fed
P55C35L10, and it was greater in this group than in the group
fed P55L45 (Table 4). LPL activity in WAT did not differ
among the group.
DISCUSSION
The purpose of this study was to evaluate the effects in rats
of lowering the C:L ratio of the diet in the context of a normal
or high-protein diet on energy intake, lipogenesis, and adiposity.

TABLE 2
Body weight and organ weights of rats after ad libitum consumption of the P14C76L10,
P55C35L10, or P55L45 diet for 37 d1

P14C76L10 P55C35L10 P55L45

Weight, g
Initial 205 6 5 209 6 9 211 6 3
Final 366 6 15a 359 6 27b 340 6 13c
Tissues and organs, g/100 g body weight
Liver 3.59 6 0.31b 4.02 6 0.40ab 4.25 6 0.64a
Epididymal tissue 3.21 6 0.75ab 2.58 6 0.41b 1.78 6 0.43c
Retroperitoneal tissue 3.30 6 0.67a 2.66 6 0.67ab 1.94 6 0.69b
Subcutaneous tissue 4.07 6 0.88a 3.71 6 0.68a 2.75 6 0.64b
WAT 10.57 6 2.02a 8.96 6 1.16a 6.46 6 1.61b
SBAT 0.33 6 0.03a 0.28 6 0.05b 0.22 6 0.04c
Stripped carcass 39.22 6 1.55b 40.64 6 1.86b 42.67 6 1.88a
AI, g/100 g stripped carcass 27.1 6 6.0a 22.3 6 4.2b 15.1 6 4.0c

1 Values are means 6 SD, n ¼ 8. Means in a row without a common letter differ, P , 0.05.

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 CARBOHYDRATE-FREE DIET AND ADIPOSITY
FIGURE 2 Adipocyte size distribution of retroperitoneal (A) and
subcutaneous adipose tissue (B) of rats consuming the P14C76L10,
P55C35L10, or P55L45 diet ad libitum. Values are means 6 SD, n ¼ 8.
Distributions without a different letter differ P , 0.05.
Lowering the C:L ratio reduced energy intake, body weight
gain, liver lipogenesis, adipocyte size, and fat deposition in
adipose tissue, but did not modify the number of adipocytes,
except in the case of the carbohydrate-free diet.
As shown previously, compared with a high-carbohydrate,
normal-protein, low-fat diet (P14C76L10), rats fed a high-
protein, low-fat diet (P55C35L10 group) (obtained by replacing
protein for carbohydrate) had reduced energy intake (4).
Indeed, proteins were shown previously to have the strongest
appetite suppressive effect of the 3 macronutrients in animals
and humans (14). This could explain the differences in food
TABLE 3
Cellularity of retroperitoneal and subcutaneous adipose tissues of rats after ad libitum
consumption of the P14C76L10, P55C35L10, or P55L45 diet for 37 d1
Retroperitoneal adipose tissue
Adipocyte size, mm
Adipocyte density, cells/mg tissue
Adipocytes in retroperitoneal adipose tissue, n
Subcutaneous adipose tissue
Adipocyte size, mm
Adipocyte density, cells/mg tissue
Adipocytes in subcutaneous adipose tissue, n
1 Values are means 6 SD, n ¼ 8. Means in a row without a common letter differ, P , 0.05.
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1259
intake between the P14C76L10 group on the one hand and the
P55C35L10 and P55L45 groups on the other hand. For weight
gain and adiposity, the differences between groups could be due
to several mechanisms that are interdependent. First, the diver-
sion of amino acids to catabolic pathways and gluconeogenesis
is thought to be associated with a stronger thermogenic effect of
the diet, which induces a reduction in food efficiency, and
consequently weight gain and adiposity (15). Indeed, higher
food efficiency leads to fat storage. That could explain why rats
fed P55C35L10 had a lower weight gain and less adiposity than
the P14C56L30 group. To a further extent, the lower weight
gain in the P55L45 group compared with the P55C35L10
group, despite their similar food intake, could also be due to a
subsequent increase in gluconeogenic activity to compensate
for the absence of carbohydrate from the diet. These results
show that in fact, the carbohydrate content in the diet, which
determines the level of gluconeogenic activity, is directly
responsible for the food efficiency of the diet. Consequently, the
carbohydrate content of the diet is correlated directly with
weight gain and adiposity. Second, carbohydrate is the main
secretagogue of insulin, which stimulates fat storage. If lipid is
associated with a high carbohydrate content, which is the case
for the P14C76L10 diet, it leads to the development of adiposity.
On the contrary, in the absence of carbohydrate in the diet
(P55L45 group), insulinemia remains low, and lipid oxidation is
increased at the expense of lipid deposition. Consequently, the
lowest C:L ratio (C:L ¼ 0), induced the lowest weight gain, fat
body mass, adipocyte size, total adipocyte number, and fatty acid
synthase activity. These results agree with those of Klein and
Wolfe (16), indicating that the carbohydrate level of the diet
appears to be the most important factor in driving lipid
metabolism. Third, the reduction in liver FAS produced by
reducing the C:L ratio was in line with previous observations
(17,18). Indeed, genes coding for fatty acid synthesis enzymes are
activated in response to a high carbohydrate content in the diet
(19,20). In rats fed the control diet with a high carbohydrate
content, some of the glucose-derived acetyl CoA was used as a
precursor for fatty acid synthesis. Decreasing the carbohydrate
content in the diet led to a reduction in FAS expression and
activity, and in the flux of glucose to fatty acid synthesis, which
subsequently reduced adipocyte size and fat mass.
In contrast, the precise origin of the reduction in the number
of adipocytes, which was specific to the P55L45 diet and was
not observed with the P55C35L10 diet, remains to be de-
termined. It likely originated in part from the more efficient
downregulation of FAS and fatty acid synthesis in the liver,
inducing a decrease in fatty acid availability in adipose tissue
that ultimately reduced adipocyte differentiation. Other spe-
P14C76L10 P55C35L10 P55L45
111.1 6 10.1a 104.2 6 3.0b 101.2 6 4.4b
1133 6 131c 1344 6 151b 1504 6 133a
14.05 6 3.24a 13.61 6 4.05a 9.55 6 3.82b
111.6 6 2.8a 104.4 6 2.4b 101.0 6 3.9b
1087 6 88b 1269 6 87b 1516 6 181a
13.36 6 3.03a 15.09 6 3.84a 12.65 6 2.72a
 1260 PICHON ET AL.
TABLE 4
Lipolysis and lipogenesis in liver and in retroperitoneal WAT,
respectively, of rats after ad libitum consumption of the
P14C76L10, P55C35L10, or P55L45 diet for 37 d1
P14C76L10 P55C35L10 P55L45
Fatty acid synthase 0.30 6 0.09a 0.21 6 0.07b 0.10 6 0.04c
activity, [NADPH,H1]
nmol/(mg liver proteins)
Lipoprotein lipase activity, 91.1 6 50.8 82.8 6 35.5 137.7 6 99.3
nmol fatty acid
formed/(h10000 cells)
1 Values are means 6 SD, n ¼ 8. Means in a row without a common
letter differ, P , 0.05.
cific effects of amino acids and fatty acids on adipose tissue dif-
ferentiation may also be involved.
In conclusion, the present study confirms the appetite
suppressive effect of protein and reinforces the idea of the
crucial role of the carbohydrate:fat ratio in the control of
lipid metabolism. As observed previously, increasing the
amount of protein in the diet, in the context of a low or high
fat content, decreased energy intake more than a high-
carbohydrate, normal-protein diet. In addition, a high-protein,
high-fat, carbohydrate-free diet did not further modify energy
intake. As a consequence, the reduction in food intake is a
specific effect of protein. This study also demonstrated the
major role of the carbohydrate:lipid ratio of the diet. First,
glucose is the main secretagogue of insulin, and consequently
stimulates energy storage, particularly fat storage. Furthermore,
carbohydrates activate genes coding for FAS, which also
contribute to fat storage. Finally, lowering the carbohydrate
content of the diet enhances gluconeogenic activity and
consequently reduces energy storage as fat storage because of
the high energetic cost of gluconeogenesis. Extrapolation of
these results to humans requires additional study because de
novo lipogenesis is thought to be less marked in humans than in
rats. Nevertheless, it seems that high-fat, carbohydrate-free
diets are able to enhance nutritional status by reducing fatty
acid de novo synthesis and fat storage. Moreover, these diets,
although unusual, meet nutritional requirements because
dietary carbohydrates are not indispensable and their absence
can be compensated by gluconeogenesis.
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