PAPER Childhood obesity and insulin resistance in a ... - Nature

International Journal of Obesity (2005) 29, 324–333 & 2005 Nature Publishing Group All rights reserved 0307-0565/05 $30.00 www.nature.com/ijo

PAPER Childhood obesity and insulin resistance in a Yucatan mini-piglet model: putative roles of IGF-1 and muscle PPARs in adipose tissue activity and development SP Se´bert1, G Lecannu1, F Kozlowski1, B Siliart2, JM Bard3,4, M Krempf4 and MM-J Champ1,4* 1

Unite´ des Fonctions Digestives et de Nutrition Humaine, Institut National de la Recherche Agronomique (INRA), Nantes, France; 2Laboratoire des Dosages Hormonaux, Ecole Nationale Ve´te´rinaire de Nantes, Nantes, France; 3Faculte´ de Pharmacie, Laboratoire de Biochimie, Nantes, France; and 4Centre de Recherche en Nutrition Humaine de Nantes, INSERM U 539, CHU de Nantes, Nantes, France OBJECTIVE: To explore metabolic and cellular modifications induced during childhood obesity, in a novel animal model of obese mini-piglets. DESIGN: A total of 10 four-month old Yucatan mini-pigs were followed from prepuberty to adulthood. Animals were divided into two groups. The first one had been overfed (OF) a western-type diet and the second one had been normally fed a control recommended human-type diet (NF). MEASUREMENTS: Plasma insulin-like growth factor 1 (IGF-1), insulin, leptin, nonesterified fatty acids, triglycerides (TGs) and glucose were determined at sexual maturity and at young adulthood. Quantitative gene expressions of peroxysome-proliferatoractivated receptors (PPARs), glucose transporter 4, insulin receptor, IGF-1, leptin and interleukin-6 (IL-6) in skeletal muscle, adipose tissue and liver were also measured at both stages. Adult insulin sensitivity was measured via euglycaemic– hyperinsulinaemic clamps. RESULTS: Increased body weight in adult OF pigs was associated with increased body size and low insulin sensitivity. Sexually mature OF pigs had higher IGF-1 plasma concentrations than their lean littermates (Po0.05). In the OF group, TGs and glucose were both decreased (Po0.05). Muscle PPARg and a in OF pubescent pigs as compared to NF pigs were 11 times higher and 20 times lower, respectively (Po0.01). CONCLUSION: Obesity and insulin resistance induced by overfeeding mini-pigs during development and puberty were not associated with the cluster of metabolic modifications frequently observed in their adult littermates. Increased IGF-1 concentrations and modifications of skeletal muscle PPAR (a and g) expressions may help the young obese pig to partially regulate its glycaemia and triglyceridaemia through an increase of fat mass, which maintains its high insulin sensitivity. International Journal of Obesity (2005) 29, 324–333. doi:10.1038/sj.ijo.0802823 Published online 11 January 2005 Keywords: mini-pigs; insulin resistance; IGF-1; PPAR

Introduction The prevalence of obesity in children and adolescents is rising dramatically throughout the world. In the US1 and in Northern Europe,2,3 children and adolescents are becoming bigger than ever and the prevalence of childhood obesity is sometimes higher than that of adults.4

*Correspondence: Dr MM-J Champ, INRA., Unite´ Fonctions Digestives et ˜re, BP 71627, 44316 Nantes cedex Nutrition Humaine, rue de la Ge´raudie 03, France. E-mail: [email protected] Received 4 February 2004; revised 31 August 2004; accepted 22 September 2004; published online 11 January 2005

Obesity, and especially central obesity in adulthood, represents an independent risk factor for chronic diseases such as type 2 diabetes,5 cardiovascular diseases6 and even hypertension.7 They are linked to a cluster of risk factors, known as the metabolic or insulin-resistance syndrome, and include hyperinsulinaemia, high triglyceride (TG) levels, elevated free fatty acids and insulin resistance.8,9 Cellular effectors involved in glucose and lipid metabolism, insulin signalling, inflammation or even cell proliferation and differentiation are also affected by obesity. Indeed, gene transcriptions of glucose transporter (GLUT) 4, insulin receptors (INSRs), leptin, interleukin-6 (IL-6) or peroxysome proliferator-activated receptor (PPAR) a and g are modified

Childhood obesity and insulin resistance in a Yucatan mini-piglet model SP Se´bert et al

325 and may partially explain some of the risk factors associated with obesity in humans and in a wide range of animal models.10,11 Such a risk factor cluster in the case of metabolic syndrome could be associated with childhood obesity. Furthermore, even if few cohort studies have revealed hyperinsulinaemia or higher TG levels associated with high body mass index in children and adolescents,12–15 other authors have observed conflicting results16,17 and nothing is known about gene regulation in obese children and adolescents. Moreover, during sexual maturation, growth factors rise dramatically and may modify insulin sensitivity and its consequences.18,19 Insulin-like growth factor I (IGF-1) may be involved in the phenomenon. The liver provides the majority of circulating IGF-1 but muscle and adipose tissues also contribute to IGF-1 serum levels.20 Besides promoting growth, IGF-1 also displays a wide range of metabolic effects that mimic insulin in glucose and lipid metabolism.21 The precise role of IGF-1 in insulin sensitivity and obesity is still unclear. Indeed, low22 and high IGF-1 serum levels have been associated with insulin sensitivity in animal models and humans.23,24 Studies of the evolution of body weight with obesity, its cluster of common risk factors and IGF-1 (plasma concentrations and synthesis) during sexual maturation would be helpful to understand childhood obesity and its consequences. This would involve longitudinal studies with a strict control of food intake from childhood to adulthood. To provide an alternative to such long and difficult experiments, we are developing a mini-pig model with obesity induced before sexual maturation. Adult mini-pigs have been widely used,25,26 their omnivorous diet, their cardiovascular system that is quite similar to humans, their human-like physiology and physiopathology related to obesity and type 2 diabetes and their large size are very useful to mimic obese humans. The objective of the present study was to characterise metabolic and cellular modifications associated with obesity, IGF-1 and insulin sensitivity in a young mini-pig in order to propose a model of obesity acquired during sexual maturation.

recommended caloric intake for pigs27) a western-type diet, rich in saturated fats (50% of total fats) and in high glycaemic index carbohydrates. In the second group, which served as a control, mini-pigs were fed a recommended human-type diet, rich in monounsaturated fats (50% of total fats) and in low glycaemic index carbohydrates. The amount of food distributed to the control group was based on the nutritional recommendation for mini-pigs.27 In this study, the first group was referred to as OF (for overfeeding) and the second was referred to as NF (normal feeding). This study was conducted from pig ‘childhood’ (4 months old71 week) to adulthood (16 months old71 week) with the first 6 months’ period studied corresponding to the minipigs’ sexual maturation (sexual maturity: between 7and 10 months of age). Pigs were fed twice a day: the OF diet provided 435 kcal BW0.75 24 h1 and the NF diet provided 290 kcal BW0.75 24 h1. The amount of food intake was controlled after each meal for every mini-pig. During sexual maturation, the amount of food distributed to each group was weekly adjusted. After sexual maturity (10 months old), the amount of food distributed to pigs was maintained constant. Throughout the entire study, food distribution for OF pigs was calculated on the basis of the mean metabolic weight (BW0.75) of NF pigs. Food distribution to the NF group was calculated on the basis of their individual body weight (Table 1). We compared three different stages: baseline (4 months old71 week), sexual maturity (10 months old71 week) and adulthood (16 months old71 week). Individual body weight was measured each week. Anthropometric parameters such as size and abdominal and neck circumferences were measured at sexual maturity. We defined a porcine obesity index (POI) on the basis of these three last parameters. The pig body is considered to be like a truncated cone where the base is represented by the abdomen (A), the top by the neck (N) and the length by the body size (BS). POI was defined (l/cm) as: Volume ðlÞ ¼ ðpðBS=3Þ ðcmÞfðAÞ2 ðcmÞ þ ðNÞ2 ðcmÞ þ ðA ðcmÞN ðcmÞÞgÞ103

Materials and methods Animals Noncastrated male Yucatan mini-pigs (n ¼ 10) (Yucatan micropigs, IFFA-Credo-Charles River, Dardilly, France), 4-month old, were used in this study. Before experiments, mini-pigs were acclimated to their local environment for 2 weeks: natural light/dark cycle, temperature-controlled room (20241C), individual feeding boxes and free access to water. All experimental treatments were in accordance with French legislation on animal experimentation (Decree 87–848 of the French penal code, 1987). Experimental design Animals were divided into two identical groups of five animals. A group of mini-pigs were overfed (1.5 times the

where BS is the body size and A and N are the radius of the abdomen (A) and the neck (N). It was then possible to determine an index in litre per centimeter: Porcine obesity index ðPOIÞ ðl=cmÞ ¼ V ðlÞ=BS ðcmÞ At baseline, sexual maturity and adulthood, we collected venous blood samples in tubes containing heparin to determine TG, glucose, insulin, leptin and IGF-1 plasma concentrations. Plasma nonesterified fatty acids (NEFA) were measured in tubes containing EDTA. Insulin sensitivity was determined at adulthood with euglycaemic–hyperinsulinaemic clamps. At sexual maturity and at adulthood, liver, skeletal muscle and adipose tissues were sampled to measure gene expressions. At the end of the study, the pigs were killed with sodium pentobarbital. International Journal of Obesity


326 Table 1

Composition of NF (normal feeding) and OF (overfeeding) diets NF diet

OF diet

290

435

50 90 10

53 90 10

35 30 50 20 9.2

37 50 30 20 0

15

10

3.58 F F 70.75

F 5.39 71.22 F

F 3.05 F 11.53 1.19

12.91 0.29 4.19 F F

Casein

6.56

3.50

Vitamin mixa Mineral mixa Antioxidant (butyl-hydroxy toluene)a

2.15 1.08 0.10

0.80 1.60 0.09

Energy intake (kcal BW0.75 day1) Energy distribution (% of total energy) Carbohydrates Starch Sugar Fat SFA MUFA PUFA n-6/n-3 Proteina 1

Nutrient (g 100 g DM ) Sucrose Glucose Dry instant mashed potatoes Ground wheat (whole grain) Butter Coconut oil Sunflower margarine Olive oil Soybean oil

a Vitamins, minerals, antioxidant and proteins have been reduced in the OF diet to provide the same amount (g kg1) as for NF pigs. BW, body weight; DM, dry matter; SFA, saturated fatty acids; MUFA, monounsaturated FA; PUFA, polyunsaturated FA.

Analytical procedures Fasted plasma analyses. TGs were determined with the Triglycerides PAP 1000 kit (BioMerieux, Lyon, France). Plasma glucose concentrations were measured colorimetrically (glucose RTU, BioMerieux, Lyon, France). NEFA were immediately determined from EDTA plasma samples (NEFAC Wako Chemical GmbH, Aix en Provence, France). Insulin was determined by radioimmuno assay (Insik 5, Diasorin, Vercely, Italy). Leptin determination was made with a multispecies kit (Multi-Species Leptin RIA Kit, LINCO Research, Inc., USA). Plasma IGF-1 concentrations were determined by using a radioimmuno assay (Nichols Institute Diagnostics, CA, USA).

Determination of insulin sensitivity Surgery. At adulthood, pigs were surgically fitted out with catheters to perform euglycaemic–hyperinsulinaemic clamps. Anaesthesia was induced after an overnight fast, by intramuscular injection of ketamine (10.0 mg kg1) and was International Journal of Obesity

maintained by inhalation of 1–2% isoflurane in oxygen (4 l min1) and nitrogen monoxide (4 l min1). Polyurethane catheters (Tygon Norton, USA) were implanted in the jugular vein and the carotid artery. Catheters were kept patent by daily flushing with NaCl and heparin. Euglycaemic–hyperinsulinaemic clamps. Adult insulin sensitivity was assessed using the euglycaemic clamp technique with a continuous infusion of insulin (Actrapids, human insulin, Novo Nordisk, Bagsvaerd, Denmark) through a jugular vein (infusion rate: 2 mU kg1 min1). Euglycaemia was maintained with a perfusion of glucose solution (30%, Laboratoire Aguettant, Lyon, France), enriched with dipotassic phosphate (Laboratoire Aguettant, Lyon, France).28 The glucose infusion rate (GIR) in mg kg1 min1 determined at equilibrium was a measurement of whole body insulin sensitivity. Suppression of hepatic glucose production was measured via the clamp using a primed constant (6, 6 D2 Glucose).29

Adipose tissue, skeletal muscle and liver gene expressions At sexual maturity and adulthood, visceral (intra-abdominal) (VAT) and dorsal subcutaneous adipose tissue (SAT), skeletal muscle (abdominal muscle) and liver were sampled under RNase-free conditions for quantitative reverse transcriptionpolymerase chain reaction (RT-PCR). As the specificity of adipose tissues were characterised by a small amount of RNA (10 mg RNA extracted from 100 mg tissue) and a large amount of fat (90%),30 we had to adapt methods to provide a sufficient amount of total RNA. Briefly, approximately 100 mg of adipose tissue (visceral or subcutaneous) was homogenised in 4 ml of QIAzols lysis reagent (Qiagen S.A., Courtaboeuf, France). Homogenates were incubated in a 371C water-bath for 2 min and vigorously shaken for 30 s. Fat was discarded after centrifugation of the homogenates for 5 min at 2500 g at room temperature. RNA was collected just below the lipid supernatant and was extracted as described in the RNeasys lipid tissue mini-kit protocol (Qiagen S.A., Courtaboeuf, France). Total RNA from liver and skeletal muscle was extracted according to the Chomzinski and Sacchi31 method by using TRIzols reagent (Invitrogen, Cergy Pontoise, France). DNase treatments (RQ1 Dnase, Promega, Charbonniere, France) were performed on each extract to eliminate DNA traces and RT was performed with 2 mg of total RNA with superscript IIs reverse transcriptase (Invitrogen, Cergy Pontoise, France). Quantitative gene expressions were measured with a quantitative thermocycler (Biorad, Marnes-La-Coquette, France) where cDNA amplification was detected with the Quantitecs SYBR green PCR kit (Qiagen S.A., Courtaboeuf, France). Reactions were compared with the 18S ribosomal RNA or glyceraldehyde phosphate deshydrogenase (GAPDH). GLUT 4, INSR, IL-6, leptin (Ob), IGF-1 and PPARg gene expressions were measured in adipose tissue. GLUT 4, INSR, PPAR (a and g)


327 and IGF-1 mRNA content were measured in skeletal muscle, whereas only IGF-1 gene expression was measured in liver. Gene expression was measured by using the 2qCT method where qCT was the cycle threshold (CT) difference between housekeeping genes and a defined gene in each sample determined at an arbitrary threshold established at 25. The gene expressions were expressed as relative values. We took a reference (1.00) for each gene, represented by the mean expression of a gene at sexual maturity in the NF pig group. To compare gene expressions of visceral and subcutaneous adipose tissue, we took the mean NF pig group value of each gene in the VAT at sexual maturity as a reference.

Statistical analysis Data have been reported as mean values7s.e.m. Statistical analyses were made with Staviews software to perform Student paired t-tests where a P-value o0.05 between groups represents significant differences.

Results

Figure 1 Body weight evolutions (a), anthropometric parameters (b) and porcine obesity index (c) in NF and OF mini-pigs. *Po0.05 between groups.

Anthropometric parameters Initial body weights were identical in both groups. At the end of sexual maturation, NF and OF mini-pigs weighed 3372.6 and 5776.8 kg (Po0.0001) and 5179.4 and 107714.6 kg at adulthood (Po0.0001), respectively (Figure 1a). Sexually mature NF mini-pigs were smaller than OF animals and their abdominal and neck circumferences were both lower as compared to OF mini-pigs (Figure 1b). Average POI measured after sexual maturation was approximately two times lower in NF than in OF mini-pigs (0.4770.04 vs 0.9770.11. l cm1) (Figure 1c).

Plasma measurements and insulin sensitivity In Figure 2a and b, the different evolutions of TG and glucose concentrations between groups are shown. In the NF group, TG and glucose concentrations were not modified during the whole study. TG concentrations in OF pigs decreased from baseline to adulthood (Po0.05). In this group, fasting glucose concentrations decreased from baseline to the end of sexual maturation (Po0.05) and remained stable and significantly lower than baseline concentrations at adulthood (Po0.05). Comparisons between groups showed higher TG plasma concentrations in NF sexually mature mini-pigs than in OF animals and higher plasma glucose concentrations in NF animals than in OF mini-pigs at sexual maturity and adulthood (Po0.05). Results of plasma NEFA, insulin and leptin variations from baseline to adulthood are presented in Table 2. During the first 6 months, from baseline to sexual maturity, NEFA remained stable in the NF group and increased in OF minipigs (Po0.05). At adulthood, NEFA increased in the NF group and reached plasma values measured in OF pigs. In both groups, insulin increased (Po0.05) from baseline to sexual

Figure 2 Evolution of TG (a) and glucose (b) fasting plasma concentrations from baseline to adulthood in NF and OF mini-pigs. *Po0.05 between baseline concentrations in a group; $Po0.05 between groups at the same time.

International Journal of Obesity


328 maturity and remained stable until adulthood. Leptin increased significantly in both groups throughout the entire experimental period but no significant differences were observed between NF and OF mini-pigs. Mean (7s.e.m.) glucose infusion rates and plasma insulin levels measured using euglycaemic–hyperinsulinaemic clamps are shown in Table 3. We measured a glucose infusion rate (GIR) that was twice as high in adult NF mini-pigs as in adult OF mini-pigs (P ¼ 0.003) with identical insulin levels between groups. Those results revealed higher insulin sensitivity in NF pigs as compared to OF mini-pigs.

Skeletal muscles and adipose tissue gene expressions GLUT 4, INSR, PPARa and PPARg gene expressions in abdominal skeletal muscle are shown in Table 4. Sexually mature NF mini-pigs had lower expressions of PPARg gene

Table 2 Comparisons of plasma concentrations (mean values7s.e.m.) at baseline, after reaching sexual maturity and adulthood in NF and OF mini-pigs

NEFA (mmol l1) NF OF Insulin (mU ml1) NF OF Leptin (ng ml1) NF OF

Baselinea

Sexual maturityb

Adulthood c

0.1370.07 0.1170.05

0.1970.06 0.3270.07*,**

0.2870.08* 0.3170.09*

15.271.80 17.075.20

28.877.82* 24.877.80*

27.273.40* 27.074.00*

1.2071.12 1.0070.50

4.6072.32* 2.8370.89

3.8071.28* 10.078.00*

*Po0.05 vs baseline in a group. **Po0.05 vs group. aBaseline: 4-month old animals. bSexual maturity: 10-month old animals. cAdulthood: 16-month old animals.

Table 3 Comparisons of euglycaemic–hyperinsulinaemic clamp data after 12 months of experimental diets between NF and OF adult mini-pigs

1 a

Glycaemia (mmol l ) Insulinaemia (mU ml1)a GIR (mg kg1min1)

NF

OF

P

4.7670.41 227714.2 14.4672.23

4.2770.11 231714.5 6.4170.57

NS NS o0.01

a

Glycaemia and insulinaemia at equilibrium. GIR: glucose infusion rate.

Table 4

(Po0.01) and higher transcriptions of PPARa mRNA (Po0.01) than OF mini-pigs. At that time, GLUT 4 and INSR gene expressions were identical in both groups. Gene expression of GLUT 4 in skeletal muscles of adult NF minipigs was higher than in OF animals. Genes encoding for INSR and PPARa were more expressed in adult NF animals as compared to adult OF mini-pigs with a borderline significance (P ¼ 0.06). Between the end of sexual maturation and adulthood, GLUT 4 and INSR gene transcriptions increased in NF pigs (Po0.05) and remained stable in OF mini-pigs. PPAR (a and g) gene expressions from sexual maturity to adulthood were stable in the NF group. As compared to sexual maturity, PPARg decreased and PPARa increased in OF adult mini-pigs (Po0.05). Table 5 represents gene expression evolutions measured in visceral and subcutaneous adipose tissues in NF and OF minipigs. At sexual maturity, only leptin in VAT was significantly different between groups. This gene was 22.8 times less expressed in NF than in OF mini-pigs (Po0.01). Sexually mature NF and OF mini-pig gene transcriptions for GLUT 4 and INSR were higher in VAT than in SAT (Po0.05). Leptin gene expression was higher in VAT than in SAT in OF minipigs at the end of sexual maturation (Po0.05). At adulthood, we measured lower gene expressions of VAT GLUT 4 (P ¼ 0.06) and leptin (Po0.05) in NF than in OF mini-pigs. The transcriptions of genes encoding for GLUT 4, IL-6 and leptin were significantly lower in the SAT of NF than in the SAT of OF mini-pigs (Po0.05). In adult NF mini-pigs, PPARg gene expression was higher in VAT than in SAT (Po0.05) and, in OF, only leptin gene expression was higher in VAT than in SAT (Po0.05). In NF mini-pigs, VAT gene expressions were stable from sexual maturity to adulthood. GLUT 4, INSR, PPARg and leptin gene transcriptions were significantly increased between the end of sexual maturation and adulthood in NF mini-pigs. OF gene expression in VAT for INSR decreased between sexual maturity and adulthood (Po0.05). GLUT 4, PPARg and IL-6 gene expressions significantly increased between sexual maturity and adulthood in the SAT of OF pigs (Po0.05).

IGF-1 plasma concentrations and gene expressions Figure 3 shows the evolution of fasting insulin-like growth factor I plasma concentrations from baseline to adulthood in mini-pig groups. Baseline IGF-1 plasma levels were identical in NF and OF groups (P40.05). IGF-1 plasma levels in the NF

Comparison of relative gene expressions measured in NF and OF mini-pig skeletal muscle (abdominal muscle) at sexual maturity and at adulthood Sexual maturity

GLUT 4 INS R PPARg PPARa

Adulthood

Age

NF

OF

P

NF

OF

P

NF

OF

1.0070.33 1.0070.37 1.0070.36 1.0070.46

0.9070.55 0.8770.39 11.874.64 0.0570.03

NS NS o0.01 o0.01

1.5170.39 2.9470.54 0.6070.23 2.8970.59

0.9170.21 1.6270.83 0.3570.20 1.5870.74

0.05 0.06 NS 0.06

0.05 0.05 NS NS

NS NS o0.05 o0.05



329 Table 5 Comparison of relative gene expressions measured in visceral (VAT) and subcutaneous (SAT) adipose tissues of NF and OF mini-pigs at sexual maturity and at adulthood Sexual maturity

Adulthood

Age

NF

OF

P

NF

OF

P

NF

OF

Visceral adipose tissue GLUT 4 INS R PPARg IL-6 Leptin

1.0070.35 1.0070.25 1.0070.13 1.0070.41 1.0075.30

1.5870.44 1.1670.41 1.2270.29 4.6474.47 22.873.17

NS NS NS NS o0.05

0.6470.18 0.8770.14 1.0970.08 1.3771.04 11.174.82

1.7370.60 0.5970.16 0.9770.28 3.2771.27 29.576.67

0.06 NS NS NS o0.05

NS NS NS NS NS

NS o0.05 NS NS NS

Subcutaneous adipose tissue GLUT 4 INS R PPARg IL-6 Leptin

0.2470.08 0.2570.24 0.0870.08 0.1670.13 0.2570.40

0.3270.18 0.4770.37 0.1870.14 0.2670.25 0.4771.40

NS NS NS NS NS

0.7970.36 0.8070.28 0.6170.27 0.2570.08 4.5172.76

1.6370.46 0.8070.26 0.8670.40 1.9071.20 9.2772.93

o0.05 NS NS o0.05 o0.05

o0.05 o0.05 o0.05 NS o0.05

o0.05 NS o0.05 o0.05 NS

o0.05 NS o0.05 NS NS

o0.05 NS o0.05 NS o0.05

NS NS o0.05 NS NS

NS NS NS NS o0.05

Visceral vs subcutaneous GLUT 4 INS R PPARg IL-6 Leptin

(Po0.05). VAT and liver IGF-1 gene expressions were statistically identical between groups. Concerning this time point, in the NF group, the IGF-1 gene was more expressed in VAT than in SAT (Po0.05). At adulthood, the transcription of IGF-1 mRNA was lower in SAT and higher in the muscles of NF pigs than in those of OF mini-pigs. In NF pigs, IGF-1 gene expression increased in SAT and skeletal muscle from sexual maturity to adulthood (Po0.05). In OF mini-pigs, SAT IGF-1 mRNA content increased (Po0.05) and decreased in skeletal muscles (Po0.05) between sexual maturity and adulthood.

Figure 3 Evolution of fasting plasma concentrations of IGF-1 from baseline to adulthood in NF and OF mini-pigs. *Po0.05 between baseline concentrations in a group; $Po0.05 between groups at the same time.

group increased from baseline to adulthood but only adulthood mean IGF-1 concentrations were significantly higher as compared to baseline (Po0.05). OF mini-pig IGF-1 concentrations increased from baseline to sexual maturity (Po0.01) and decreased from sexual maturity to adulthood (Po0.05). At the end of sexual maturation, NF mini-pigs had lower IGF-1 concentrations than OF mini-pigs (186760 vs 337757 ng ml1; P ¼ 0.02). No significant differences were observed between adult groups. Table 6 represents relative IGF-1 gene expressions in adipose tissues (VAT and SAT), skeletal muscle and liver. At sexual maturity, we measured lower IGF-1 gene expression in NF mini-pigs than in OF animals in SAT and skeletal muscle

Discussion This study was designed to provide a model of childhood obesity by overfeeding mini-piglets with a western-type diet and exploring the associated parameters. Numerous similar experiments have been carried out on adult animal models but, to our knowledge, none have been carried out on immature animals. As compared to previous experiments conducted in postsexually mature mini-pigs,32,33 the OF treatment dramatically increased body weight. Moreover, as previously described in adolescents,34,35 OF mini-pigs were taller than NF mini-pigs. Obesity and insulin resistance in adult humans and adult animal models are frequently linked to high TG, glucose, insulin, NEFA and leptin plasma concentrations.36–39 In the present study, insulin resistance was only measurable by performing euglycaemic–hyperinsulinaemic clamps. Contrary to studies performed on adults, fasting glucose and TG plasma concentrations decreased in International Journal of Obesity


330 Table 6 IGF-1 relative gene expressions measured in NF and OF mini-pigs in visceral (VAT) and subcutaneous (SAT) adipose tissues, skeletal muscle (abdominal muscles) and liver at sexual maturity and at adulthood Sexual maturity

Adulthood

Age

NF

OF

P

NF

OF

P

NF

OF

VAT SAT

1.0070.22 0.1770.13 o0.05

1.4370.43 0.9570.78 NS

NS o0.05

0.7870.17 1.4370.40 o0.05

1.3770.30 3.5171.39 o0.05

NS o0.05

NS o0.05

NS o0.05

Skeletal muscle

1.0070.41

2.6571.20

o0.05

5.0071.75

1.8570.90

o0.01

o0.01

NS

Liver

1.0070.56

1.7770.74

NS

0.4070.08

0.7670.44

NS

NS

NS

the mini-pigs that were made obese during sexual maturation. At a gene transcription level, OF pig insulin resistance was supported by classical modifications of gene expressions in adult skeletal muscles. Indeed, GLUT 4, INSR and PPARa gene expressions were lower in adult OF as compared to NF mini-pigs, suggesting lower muscle insulin sensitivity.11,40 However, GLUT 4 transcription increased in subcutaneous adipose tissue in OF mini-pigs and may indicate a higher stimulation of glucose uptake and de novo lipogenesis40 in the OF group as compared to NF animals. This difference may lead to a decrease of both plasma glucose and TG concentrations. When obesity was induced by a high glycaemic index diet in rats, Pawlak et al41 observed a drop in TGs. They suggested the possibility that fat mass growth induced higher TG capture and a more efficient lipogenesis. In our study, we hypothesised that a considerable expansion of a highly sensitive adipose tissue induced during sexual maturation may conceal metabolic consequences of insulin resistance. This hypothesis may be partially supported by PPAR (a and g) gene expression profiles in sexually mature muscles, IGF-1 plasma concentrations and IGF-1 gene expressions. Skeletal muscle PPARa and gene expression profiles differed between sexually mature mini-pigs and may indicate two different lipid utilisations. Indeed, PPARa is preferentially expressed in tissues with a high lipid catabolism (liver, small intestine, skeletal muscles, etc.)42 and PPARg in those with high anabolism of lipids, especially adipose tissues.43 The role of PPARs in skeletal muscles is still unclear. PPARa is associated with lipid oxidation,44 whereas PPARg appears to induce genes involved in lipid transport.45 In sexually mature OF pigs, higher PPARg expression may indicate a higher capture of lipid by muscles. Two hypotheses can be proposed to explain this evolution of PPARg in OF pigs. On the one hand, since PPARg is not recognised as a major site of PPARg transcription,43 it is then possible that RNA extracted from the muscle could be partially contaminated by adipose tissue infiltrating the muscle. PPARg gene expression measured at sexual maturation could reflect the increase of fat deposition in the muscle rather than a specific muscular modification. Furthermore, when comparing mRNA effiInternational Journal of Obesity

ciency between fat and muscle,30 such ‘pollution’ should be negligible and would not interfere in the measurements. On the other hand, a high activation of PPARg in vitro induces a transdifferentiation of myoblasts in mature adipocytes.46 This is technically impossible to observe in vivo. Nevertheless, sexual maturation could be especially propitious to such transdifferentiation due to muscle growth. On the contrary, we may postulate that PPARa involved in lipid catabolism44 was normally expressed in the NF pig group to maintain the muscular phenotype. As previously mentioned, obese adolescents are frequently taller than normal weight adolescents,34,35 suggesting better IGF-1/GH axis stimulation during obesity. Similar to human adolescents,18,19 IGF-1 increased in OF mini-pigs and was associated with increased body size at sexual maturity. The biology of growth and especially growth hormone (GH) secretion is a complex programmed phenomenon. Several endocrine systems such as GH, insulin and specific binding proteins (IGF BP 1–6) control IGF-1 serum concentrations. Moreover, observations of humans and animals demonstrate that IGF-1 serum concentrations can only increase during childhood, until the end of puberty.23 This particularity of IGF-1 may explain, in spite of an identical diet before and after sexual maturation, why IGF-1 plasma differences were only observed between young mini-pigs. The interrelationship between energy intake, IGF-1 serum concentrations and growth performances has already been described in pigs,47 cattle48 and human adolescents.23 We think that an increase of the energy intake could stimulate IGF-1 synthesis and consequently induce tissue development and an increase of energy storage. Despite promoting growth and tissue differentiation,49 especially in the case of adipose tissues,50 IGF-1 also stimulates glucose51 and lipid uptake52 in muscles and adipocytes. Increased IGF-1 levels during sexual maturation could then be responsible for the increase in TG and glucose capture observed in OF mini-pigs. Nevertheless, IGF-1 serum levels decreased in adult OF mini-pigs, whereas TG and glucose remained stable and low. The transcription of IGF-1 in a tissue can reflect the level of IGF-1 protein synthesis53 and, as in the case of interleukins,54 IGF-1 has an autocrine–paracrine action.55,56 IGF-1


331 gene expression increased in adult OF SAT and may generate a high autoinduced local lipogenic environment. The association with increased GLUT 4 and leptin gene expressions in adult SAT of OF mini-pigs suggested a greater glucose and lipid uptake and supported this hypothesis.57 In obese animals, this relationship may be sufficient to maintain low TG and glucose plasma concentrations without hyperinsulinism. On the contrary, IGF-1 and GLUT 4 gene expressions decreased in adult OF skeletal muscles and may confirm the lower insulin sensitivity of this tissue as compared to NF pigs. The precise roles of IGF-1 in obesity and insulin sensitivity remain unclear. Liver IGF-1-deficient mice are insulin resistant22 and a perfusion of IGF-1 is sufficient to restore insulin sensitivity and normalise plasma parameters.22 As demonstrated in the present study, we think that IGF-1 may be partially responsible for the lack of risk factors associated with obesity and insulin resistance in OF pigs. Nevertheless, IL-6 synthesis, a well-known factor involved in insulin-resistance genesis,54 is stimulated by IGF-1.58 In normal healthy subjects (with ‘normal’ IGF-1 levels), IL-6 is a direct regulator of IGF-1 circulating levels through activation of the IGF-BP1 gene and its synthesis by the liver.59 At a higher level of IGF-1, such as serum concentrations measured in obese adolescents and in obese miniature pigs, IGF-1 through IL-6 release may induce insulin resistance in muscles.60 In conclusion, this study investigated the possible effects of obesity induced during sexual maturation in a novel animal model. As compared to adult models and humans, obesity was not associated with the usual cluster of metabolic modifications. We detected modifications in muscular PPAR (a and g) transcriptions, IGF-1 concentrations and IGF-1 transcription levels that may induce the high development of the adipose tissue and may maintain its glucose tolerance and insulin sensitivity. Unfortunately, data on obese children from childhood to adulthood are missing to confirm our observations. However, some results on insulin resistance,17 TG levels13 or IGF-1 plasma concentrations23 in childhood support our hypothesis. Nevertheless, we cannot ignore that pigs, as compared to humans, have a higher de novo lipogenesis61 that may exacerbate the phenomenon. Further studies are needed to confirm our results and to understand mechanisms linked to muscular PPARs and IGF-1 synthesis during obesity acquired before sexual maturation.

Acknowledgements We express our gratitude to D Me´sangeau and L Noah from Merck-Sante´ S.A. (Merck Sante´, Centre de Recherche de Chilly-Mazarin, Chilly-Mazarin, France) for their financial and scientific contributions and Regions Payscle la Loire and Bretagne for their Financial support. We also thank C Bonnet for his technical assistance for mRNA extractions and gene expressions and G Poupeau and JL Lescure for taking care of mini-pigs.

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