diet-induced obesity following perinatal high fat feeding in
obesity-resistant Lou/C rats
1 Preserving of postnatal leptin signaling in obesity-resistant Lou/C rats following a 1
perinatal high-fat diet 2
3
Anne-Laure Poher1, 2,Denis Arsenijevic3, Mohamed Asrih4, Abdul G. Dulloo3, François R.
4
Jornayvaz4, Françoise Rohner-Jeanrenaud1, 2 and Christelle Veyrat-Durebex1. 5
6
1 Department of Cell Physiology and Metabolism and 2Laboratory of Metabolism, Department 7
of Medicine Specialties, Faculty of Medicine, University of Geneva, Geneva, 3Department of 8
Medicine/Physiology, University of Fribourg, Fribourg, and 4Service of Endocrinology, 9
Diabetes and Metabolism, Lausanne University Hospital, Lausanne, Switzerland.
10 11
Short title: Postnatal leptin signaling in obesity resistant Lou/C rats 12
13
Corresponding author: Dr. Christelle Veyrat-Durebex 14
Department of Cell Physiology and Metabolism 15
Centre Médical Universitaire 16
1, rue Michel Servet 17
1211 Geneva-4, Switzerland 18
Tel: +41-22 379 5210 19
Fax: +41-22 379 5260 20
E-mail: christelle.veyrat-durebex@unige.ch 21
22 23 24
Manuscript Click here to download Manuscript Manuscript text and table
18042016.docx
2 Abstract
25 26
Physiological processes at adulthood, such as energy metabolism and insulin sensitivity 27
may originate before or weeks after birth. These underlie the concept of fetal and/or neonatal 28
programming of adult diseases, which is particularly relevant in the case of obesity and type 2 29
diabetes. The aim of this study was to determine the impact of a perinatal high fat diet on energy 30
metabolism and on leptin as well as insulin sensitivity, early in life and at adulthood in two 31
strains of rats presenting different susceptibilities to diet-induced obesity. The impact of a 32
perinatal high fat diet on glucose tolerance and diet-induced obesity was also assessed. The 33
development of glucose intolerance and of increased fat mass was confirmed in the obesity-34
prone Wistar rat, even after 28 days of age. By contrast, in obesity-resistant Lou/C rats, an 35
improved early leptin signaling may be responsible for the lack of deleterious effect of the 36
perinatal high fat diet on glucose tolerance and increased adiposity in response to high fat diet 37
at adulthood.
38 39
3 Introduction
40
Several evidences suggest that environmental factors modulating physiological 41
processes at adulthood, such as energy metabolism and related insulin sensitivity may originate 42
before or weeks after birth (i.e. the perinatal period) (1). This raised the concept of fetal/neonatal 43
origin or programming of adult disease, particularly with regard to obesity and type 2 diabetes, 44
together with related cardiovascular diseases (2). Thus, the pathogenesis would not be based on 45
genetic defects, but rather on altered genetic adaptation to environmental changes during 46
development, such as those occurring during modification of the mother’s diet. Indeed, many 47
studies in rodents reported that high fat feeding by pregnant or lactating females induced 48
glucose intolerance and development of obesity in the progeny during adult life (3, 4) (for recent 49
review, see (5)). Maternal high fat feeding also significantly altered the hypothalamic 50
expression of key genes encoding for proteins involved in the regulation of food intake and 51
energy homeostasis, such as the neuropeptide Y (NPY) (6), and in many case, the 52
proopiomelanocortin (POMC) system (7, 8). Importantly, maternal high fat feeding also rapidly 53
induced leptin resistance in the arcuate nucleus and increased the susceptibility to develop diet-54
induced obesity in response to a high fat diet during adulthood (9, 10). In contrast, maternal 55
undernutrition (i.e. food restriction of 50% from embryonic day E14 to postnatal day PND21) 56
was reported to induce a brown-like phenotype of gonadal white adipocytes until 30 days of 57
age, suggesting that it may have long-term consequences on energy metabolism in adult rodents 58
(11).
59
The Lou/C rat, an inbred strain of Wistar origin (12), was described as being resistant to the 60
development of obesity with age or in response to a high fat (HF) diet, compared to Wistar rats 61
(13, 14). Such a phenotype is accompanied by a lower body fat mass (15, 16), low circulating 62
leptin levels (15-17), increased central leptin sensitivity (18) and enhanced glucose tolerance, 63
as well as insulin sensitivity compared to the Wistar group (15). Interestingly, recent data also 64
4 underlined a brown-like phenotype of some adipocytes in the inguinal white adipose tissue 65
depot in Lou/C rats (19).
66
The first aim of this study was to determine the impact of a perinatal high fat (pHF) diet on the 67
regulation of energy metabolism in 28 day-old Wistar and Lou/C rats. The second aim was to 68
investigate the overall effects of a pHF diet on the development of glucose intolerance and diet-69
induced obesity at adulthood in the two strains of animals. To address these issues, three 70
experiments were designed to study some of the metabolic effects of: 1) pHF diet (from 71
embryonic day E14 to postnatal day PND28) in Wistar and Lou/C rats from birth to day 28 72
(PND0 to PND28); 2) pHF diet in adult Wistar and Lou/C rats submitted to a standard diet; 3) 73
pHF diet in adult Wistar and Lou/C rats challenged with 5 weeks of high fat diet at adulthood.
74 75
Materials and Methods 76
77
Ethics Statement 78
All procedures were performed in accordance with and approved by the Institutional 79
ethical Committee of Animal Care in Geneva and Cantonal Veterinary Office (experiment ID 80
1034/3684/2).
81 82
Animals 83
Three month-old male and female Wistar and Lou/C rats were purchased from Charles 84
River (L’Arbresle, France) and Harlan UK Limited (Oxon, UK), respectively. They were 85
housed under controlled temperature (22°C) and lighting (light on: 07:00am-07:00pm) with 86
free access to water and to a standard (STD) laboratory diet, RM3 (SDS, Essex, UK). After one 87
week of handling, they were randomly distributed in two groups, either fed with the STD diet 88
or with a 40% high fat (HF) diet (4.31 kcal/g) (2154 KLIBA NAFAG high fat purified diet, 89
5 Provimi Kliba AG, Kaiseraugst, Switzerland) for one week only (acclimation period). One 90
week later, females were placed into individual cages and bred with males for a maximum of 7 91
days. Pregnancies were confirmed by the presence of vaginal plugs. HF-fed females were then 92
kept under HF diet from the last 7 days of pregnancy (corresponding to embryonic day E14) to 93
weaning at perinatal day 21 (PND21). HF-fed pups (males and females) were kept under HF 94
diet until PND28. After delivery, litter size was minimally adjusted to 6/8 pups/litter for Lou/C 95
and to 8 pups/litter for Wistar. For aim 1, perinatal standard- (pSD) and HF-fed (pHF) Wistar 96
and Lou/C males from 4 different breeding were euthanized at PND0, PND7, PND10, PND14, 97
PND17, PND21, and PND28. For aims 2 and 3, sexing of male and female rats was performed 98
at PND28. Both pSD and pHF males were fed for 2 months with the standard laboratory diet 99
RM1 (SDS, Essex, UK). A first cohort was studied at the age of 3 months under standard diet 100
(Aim 2). A second one was submitted to 5 weeks of high fat diet (HFD) at the age of 3 months, 101
and then studied for metabolism at 4 months (Aim 3). At the end of the experiments, rats were 102
euthanized using isoflurane anesthesia (Halocarbon Laboratories, River Edge, NJ) and rapid 103
decapitation. Blood was sampled and tissues were rapidly removed, freeze-clamped and stored 104
at –80°C.
105 106
Body composition 107
An EchoMRI-700™ quantitative nuclear magnetic resonance analyzer (Echo Medical 108
Systems, Houston, TX) was used to measure body composition (total fat and lean body mass).
109 110
Peripheral (i.p.) leptin injection 111
Intraperitoneal (i.p.) injections of human leptin (2 mg/kg; Bachem, Bubendorf, 112
Switzerland) or vehicle (NaCl 0.9%) were administered 1h before the start of the dark cycle 113
(06:00pm). The anorexigenic effect of leptin was thus determined by removing the food from 114
6 the cages just before leptin injection (06:00pm), and re-introducing pre-weighed amounts of 115
food at 7:00pm. Food intake was thereafter measured at 0.5, 1, 2, 4 and 12 hrs after lights off.
116 117
Glucose (GTT) tolerance test 118
Rats were fasted for 4hrs (09:00am to 01:00pm). A glucose load of 1.5 g/kg was 119
administeredi.p. Blood samples were collected by tail nicking for further analyses of plasma 120
glucose and insulin concentrations.
121 122
Plasma measurements 123
Plasma glucose was measured by the glucose oxidase method (Glu, Roche Diagnostics 124
GmbH, Rotkreuz, Switzerland). Plasma nonesterified fatty acids (NEFA) and triglycerides 125
(TG) were determined using NEFA C (Wako Chemicals GmbH, Neuss, Germany) and 126
BioMerieux (Marcy l’Etoile, France) commercial kits, respectively. Commercial EIAs were 127
used to measure plasma leptin (Bertin Pharma, Montigny-le-Bretonneux, France), insulin 128
(Mercodia, Uppsala, Sweden), fibroblast growth factor 1 (FGF21) (R&D systems Europe Ltd, 129
Oxon, UK), and adiponectin (AdipoGen, San Diego, CA) levels.
130 131
FAS activity 132
Measurements of fatty acid synthase (FAS) activity were determined from frozen tissues 133
by spectrophotometry, as previously described (20).
134 135
Tissue processing and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 136
Total RNAs from hypothalami were extracted using a single-step extraction with Trizol 137
reagent (Sigma-Aldrich, Buchs, Switzerland). RNA integrity was assessed by electrophoresis 138
on a 1% agarose gel, and concentration was determined by spectrophotometry. A quantity of 139
7 2.5 g of total RNA was used for RT, using random primers (Promega Corporation, Madisson, 140
WI), dNTPs (Promega), Rnasin as RNase inhibitor (Promega), and the M-MLV-RT enzyme kit 141
(Invitrogen, Basel, Switzerland). For quantitative PCR (qPCR), amplification of genes was 142
performed from 12.5 ng cDNA using the SYBR® green PCR Master Mix (Applied Biosystems, 143
Foster City, CA) and an ABI7500 machine. Primers were used at a concentration of 200 to 300 144
nM and results were normalized to the expression levels of the 36b4 housekeeping gene.
145 146
Data analyses 147
Results are expressed as mean ± SEM. Comparisons between Lou/C and Wistar rats for 148
feeding patterns and metabolic parameters were performed using two-way ANOVAs (effect of 149
strain and diet) with Bonferroni as post-hoc test (GraphPad Prism, La Jolla, CA). The evolution 150
with age of plasma hormone levels and of hypothalamic mRNA expression of neuropeptides or 151
receptors implicated in the regulation of food intake was first analyzed using repeated-measures 152
ANOVA for each group. Then three-way repeated-measures ANOVAs were applied, for the 153
factors of strain, diet and age, with age treated as a repeated measure. Statistical significance 154
was established at p<0.05. Number of animals used for all measurements and the number of 155
litters represented are provided in figure legends.
156 157
Results 158
Increased postnatal leptin signaling may prevent the occurrence of increased adiposity due to 159
perinatal high fat feeding in Lou/C rats 160
At birth, body weight (BW) was higher in STD Wistar than in STD Lou/C rats (Fig.
161
1A). The presence of high fat (HF) diet during the last week of gestation induced a significant 162
increase in the birth weight of the same amplitude in both strains (F(1, 42)=34.14, p<0.0001). At 163
weaning (PND21), the effect of the perinatal HF diet was still present on BW (F(1, 108)=243.3, 164
8 p<0.0001), being more marked in Wistar (p<0.0001) than in Lou/C rats (Fig. 1B). At PND28, 165
the difference in BW was only present in Wistar rats (p<0.0001) (Fig. 1C). Interestingly, it was 166
linked to a higher percent fat mass (F(1, 27)=16.63, p<0.001), without any change in lean mass, 167
as determined using EchoMRI (Fig. 1D).
168 169
Figure 1. Effect of a perinatal HF diet on body weight and body composition in Wistar and 170
Lou/C rats. A) Body weight (BW) at birth in grams (n=7-16). B) Body weight gain from PND0 171
to PND28 in grams (n=6-30 rats/group depending on age). C) Body weight (BW) in grams at 172
d28 (n=14-24). D) Body composition expressed as percent lean and fat mass at d28 (n=6-9).
173
Values are mean + SEM. ***p<0.001 and ****p<0.0001 using a two-way ANOVA. Fig1B: * 174
is for pSD Wistar vs. pHF Wistar, † is for pSD Lou/C vs. pHF Lou/C.
175 176
The profile of different metabolites and hormones was then analyzed from PND0 to PND28 in 177
Wistar (Fig. 2A) and Lou/C rats (Fig. 2B). At birth, no significant difference appeared between 178
pSD and pHF Wistar rats. In pHF Lou/C newborns, glycemia was lower and insulinemia was 179
higher than in pSD pups. Non-esterified fatty acids (NEFA) (p<0.05) and triglycerides (TG) 180
(p=0.0624) were lower in pHF than in pSD Lou/C pups, suggesting a healthy metabolism, 181
despite the presence of the perinatal HF diet. Of note, plasma leptin levels at birth were twice 182
higher in Lou/C than in Wistar rats. To our knowledge, this is the only time point at which such 183
a hyperleptinemia is observed in this strain.
184
Over the 4 weeks, the HF diet significantly modified the profile of plasma TG (p<0.05), insulin 185
(p<0.01) and leptin (p<0.05) levels in Wistar rats. A peak of TG and insulin was seen in pHF 186
Wistar at weaning (PND21), whereas the peak of leptin observed at PND14 in pSD rats was 187
totally blunted in pHF Wistar rats. In Lou/C animals, only plasma TG (p<0.001) and NEFA 188
(p<0.001) profiles were slightly modified by the perinatal HF diet. In this group, the high leptin 189
9 levels observed at birth were completely suppressed at d7 in the pHF pups, while they were 190
maintained until d10 in pSD animals. Such leptinemia profiles in Lou/C rats may reveal the 191
occurrence of an early postnatal increase in leptin signaling, which was only partly blunted by 192
the pHF diet.
193 194
Figure 2. Effect of a perinatal HF diet on metabolic parameters in Wistar (A) and Lou/C (B) 195
rats from PND0 to PND28. Values are mean + SEM of 6-8 animals per group. *p<0.05 and 196
***p<0.001 using one-way ANOVA on repeated measures.
197 198
Gene expression of the main hypothalamic neuropeptides, such as neuropeptide Y (NPY), 199
proopiomelanocortin (POMC), Agouti-related peptide (AgRP) or brain-derived neurotrophic 200
factor (BDNF), and one of the main melanocortin receptor the MC4R, all implicated in the 201
regulation of food intake (Fig. 3A), was analyzed in pSD and pHF Wistar and Lou/C rats from 202
PND14 to PND28. NPY is an orexigenic peptide (21), like AgRP, a specific antagonist of the 203
most relevant melanocortin receptor, the MC4R (21). BDNF is on the contrary an anorexigenic 204
peptide (22, 23), like POMC (21), the precursor of αMSH. By binding to the MC4R in the 205
paraventricular nucleus, αMSH inhibits food intake and increases energy expenditure (24). The 206
expression of the leptin receptor (Obrb) was also examined.
207
The perinatal HF diet did not significantly influence mRNA expressions in Wistar animals, 208
except for Pomc expression that was slightly increased at d14 and d17 (Fig. 3B). In contrast in 209
Lou/C rats, mRNA expression of Pomc (F(3, 22)=3.07, p<0.05), Bdnf (F(3, 22)=6.12, p<0.01) and 210
Agrp (F(3, 22)=3.08, p<0.05) were significantly modified (Fig. 3C). In pHF Lou/C rats, Bdnf 211
mRNA expression was higher at d17, whereas Pomc levels were higher at weaning (PND21).
212
Finally, Agrp mRNA levels were drastically decreased in pHF Lou/C at PND28. However, 213
since these gene differences appeared at only one time point, it cannot be excluded that a modest 214
10 shift in the timing of neurons maturation is present between the 4 cohorts observed.
215
Nevertheless at PND17, the Obrb gene expression was significantly increased in the pHF Lou/C 216
group (Fig. 3D), which might contribute to counteract the deleterious effect of the pHF diet in 217
this strain.
218 219
Figure 3. Effect of a perinatal HF diet on hypothalamic neuropeptide mRNA expression in 220
Wistar and Lou/C rats. A) Schematic representation of the main hypothalamic nuclei implicated 221
in the control of food intake and metabolism. ARC: arcuate nucleus; VMN: ventromedial 222
nucleus; DMN: dorsomedial nucleus; PVN: paraventricular nucleus. Hypothalamic mRNA 223
expression of Pomc, Bdnf and Agrp in pSD and pHF Wistar (B) and pSD and pHF Lou/C (C) 224
rats. Values are expressed as percent of pSD Wistar or Lou/C rats at PND14 (100%) and were 225
normalized with the 36b4 mRNA expression. D) Hypothalamic mRNA expression of Obrb.
226
Values are expressed as percent of pSD Wistar rats at PND17 (100%) and were normalized 227
with the 36b4 mRNA expression. Values are mean + SEM (n=3-6). *p<0.05 and ****p<0.0001 228
using one-way and two-way ANOVA.
229 230
At 3 months of age, no effect of the perinatal HF diet was seen on body weight of Wistar and 231
Lou/C rats (Table 1). The food intake was also comparable in pHF and pSD animals. However, 232
a significant increase in the percent fat mass was observed in pHF compared to pSD Wistar rats 233
(F(1, 27)=12.45, p<0.001), while no change was observed for the lean mass (Fig. 3A). When 234
analyzing different plasma parameters of energy metabolism, no significant difference appeared 235
for plasma insulin and glucose levels (Table 1). Lower NEFA and leptin concentrations were 236
detected in Lou/C vs Wistar rats, as already described (15). Furthermore and as expected, the 237
higher fat mass of pHF Wistar rats was correlated with their increased plasma leptin levels.
238 239
11 Table 1. Metabolic and hormonal parameters of adult (3 months of age) pSD and pHF Wistar 240
and Lou/C rats fed a standard diet at adulthood.
241
Wistar pSD Wistar pHF Lou/C pSD Lou/C pHF
BW (g) 335 + 5 341 + 6 256 + 4ab 264 + 5ab
Food Intake (g/d) 21.8 + 0.3 22.1 + 0.3 18.5 + 0.5ab 18.5 + 0.4ab HOMA Index 20.3 + 2.4 19.1 + 1.6 8.3 + 2.1ab 12.6 + 2.0ab Insulin (ng/mL) 2.3 + 0.3 2.2 + 0.2 2.1 + 0.2 2.1 + 0.4 Leptin (ng/mL) 3.5 + 0.3 5.4 + 0.3 a 1.8 + 0.2a 2.0 + 0.5ab Glucose (mmol/L) 8.0 + 0.7 10.3 + 1.9 6.9 + 0.7 7.1 + 0.6 NEFA (mmol/L) 1.16 + 0.02 1.09 + 0.03 0.62 + 0.09ab 0.89 + 0.05ab Adiponectin (μg/mL) 33.9 + 1.6 39.8 + 5.3 13.1 + 1.1ab 18.0 + 1.3ab FGF21 (pg/mL) 174 + 29 258 + 54 339 + 65 282 + 51 The HOMA index was calculated as [fasted glycemia (mmol/L) x fasted insulinemia 242
(mUI/L)/22.5]. ap<0.05 compared to pSD Wistar and bp<0.05 compared to pHF Wistar, using 243
the Student’s t test.
244 245
Regarding hypothalamic gene expression, significant modifications of the melanocortin 246
system were observed in Wistar rats (Fig. 4B). Indeed, the perinatal HF diet induced a 1.8-, 4.8- 247
and 3-fold increase in Pomc, Agrp and Mc4r mRNA expression, respectively. A 2.2 fold 248
increase in Obrb was also observed. However, since no difference on food intake was noticed, 249
these modifications are likely to compensate each another. In pHF Lou/C rats, only the mRNA 250
expression of Mc4r and Bdnf was significantly decreased (Fig. 4C). Once again, it seems to 251
12 have no impact on the feeding pattern. Leptin sensitivity was thereafter tested by injecting 252
human leptin (2 mg/kg), 1h before the light off. Food intake was analyzed 30 minutes, 1, 2 and 253
4 hrs later. As previously observed (18), no anorexigenic effect of leptin was observed 30 254
minutes after the lights off, whereas tendencies were observed after 2 and 4 hrs in pSD Wistar 255
and pSD Lou/C rats (Fig. 4D). However, in pHF Wistar and Lou/C rats, no effect was noticed, 256
suggesting the presence of a central leptin resistance. Since Obrb mRNA expression was 257
increased in the pHF Wistar group, such central leptin resistance could be related to decreased 258
intracellular leptin signaling, whereas a decreased activity of the BDNF system could mediate 259
this process in Lou/C rats (18).
260 261
Figure 4. Effect of a perinatal HF diet on body composition, hypothalamic neuropeptide mRNA 262
expression and leptin sensitivity in 3-month old Wistar and Lou/C rats under a standard diet.
263
A) Body composition expressed in percent lean and fat mass. B and C) Hypothalamic mRNA 264
expression of Pomc, Agrp, Mc4r, Obrb and Bdnf in Wistar and Lou/C rats. Values are expressed 265
as percent of pSD Wistar or Lou/C rats (100%) and were normalized with the 36b4 mRNA 266
expression. D) Effect of acute leptin injection (i.p., 2 mg/kg, 1h before the light off) on 4hrs-267
food intake. Values are mean + SEM (n=7-12). *p<0.05, **p<0.01 and ***p<0.001 using one-268
way and two-way ANOVA.
269 270
Perinatal high fat diet induces glucose intolerance in adult Wistar, but not in Lou/C rats 271
To further characterize the metabolic phenotype of adult rats, a GTT was performed in 272
4hr-fasted rats. As can be seen on Figure 5A, HF during the perinatal period significantly altered 273
glucose tolerance in Wistar rats at 15 and 30 min. after the glucose load (F(5, 132)=29.30, 274
p<0.0001). Moreover, the areas under the curves (AUCs) of the glycemia, 120 minutes after 275
the glucose load was significantly higher in pHF Wistar compared to pSD rats (p<0.01) (Fig.
276
13 5B). This glucose intolerance was present despite a tendency toward increased insulin release, 277
as calculated using AUCs over 60 minutes (Fig. 5C), suggesting the presence of insulin 278
resistance in peripheral tissues. No alteration of glucose tolerance or of insulin secretion was 279
observed in Lou/C rats.
280
In order to explain the occurrence of glucose intolerance in pHF Wistar rats, plasma levels of 281
adiponectin and FGF21, two modulators of insulin sensitivity (25-28), were determined.
282
However, none of these hormones was significantly modified in pHF compared to pSD rats 283
(Table 1).
284 285
Figure 5. Effect of a perinatal HF diet on glucose tolerance in 3-month old Wistar and Lou/C 286
rats under a standard diet. A) Evolution of delta glycemia (mM) at 0, 15, 30, 60 and 120 min 287
after acute glucose injection (1.5 g/kg, i.p.). B) Areas under the curves (AUCs) of glycemia 288
(mmol/L x min) over 120 minutes following the glucose load. C) Areas under the curves 289
(AUCs) of insulin (ng/mL x min) over the 60 minutes following the glucose load. Values are 290
mean + SEM (n=7-12). **p<0.01, ***p<0.001 and ****p<0.0001 using the two-way ANOVA.
291 292 293
Permissive effect of a perinatal high fat diet on the development of diet-induced obesity in adult 294
Wistar, but not in Lou/C rats 295
In this experiment, adult Wistar and Lou/C animals subjected to pSD or to pHF were 296
submitted to a high fat diet for 5 weeks at the age of 3 months. A significantly higher BW gain 297
over the 5 weeks of HFD was observed in the Wistar group (F(1, 26)=10.86, p<0.01) (Fig. 6A).
298
It was correlated with a higher caloric intake (F(1, 26)=13.89, p<0.001) (Fig. 6B) and with a 299
higher percent fat mass (F(1, 26)=28.97, p<0.0001) (Fig. 6C). Accordingly, a significant 300
stimulation of FAS activity was observed in both epididymal (eWAT) and inguinal (iWAT) 301
14 white adipose tissues (Fig. 6D). When analyzing plasma parameters, no significant difference 302
appeared between Wistar and Lou/C rats (Table 2), except for plasma leptin levels, as already 303
described (15). Thus, leptinemia was higher in the two groups of Wistar compared to Lou/C 304
rats (F(1, 26)=90.27, p<0.0001), with no difference between the pSD and pHF Wistar groups.
305 306
Figure 6. Effect of a perinatal HF diet on body weight gain, daily food intake, body 307
composition, FAS activity and hypothalamic neuropeptide mRNA expression in 4-month old 308
Wistar and Lou/C rats under a high fat diet. A) Total body weight gain over the 5 weeks of HF 309
diet. B) Mean daily (24hrs) food intake expressed in kilocalories. C) Body composition 310
expressed as percent lean and fat mass. D) FAS activity (in mU per mg of protein) in the liver, 311
epididymal (eWAT) and inguinal (iWAT) white adipose tissues. E) Hypothalamic mRNA 312
expression of Pomc, Agrp, Mc4r, Obrb and Bdnf. Values are expressed as percent of pSD Lou/C
expression of Pomc, Agrp, Mc4r, Obrb and Bdnf. Values are expressed as percent of pSD Lou/C