Original Article

International Journal of Obesity (2008) 32, 648–657; doi:10.1038/sj.ijo.0803787; published online 18 December 2007

Time course and dynamics of adipose tissue development in obese and lean Zucker rat pups

E Pouteau1, S Turner2, O Aprikian1, M Hellerstein2,3, M Moser1, C Darimont1, L B Fay1 and K Macé1

  1. 1Nestlé Research Center, Nutrition and Health Department, Vers-Chez-Les-Blanc, Lausanne, Switzerland
  2. 2KineMed Ltd, Emeryville, CA, USA
  3. 3Department of Nutritional Sciences and Toxicology, 309 Morgan Hall, University of California, Berkeley, CA, USA

Correspondence: Dr E Pouteau, Nutrition and Health Department, Nestle Research Center, Vers-Chez-Les-Blanc, Lausanne 26, 1000, Switzerland. E-mail: etienne.pouteau@rdls.nestle.com

Received 23 June 2007; Revised 8 November 2007; Accepted 13 November 2007; Published online 18 December 2007.

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Abstract

Objective:

 

To evaluate the ontogeny of adipose tissue dynamics in obese and lean Zucker rat pups, from suckling to puberty.

Methods:

 

The trial had a two-group parallel design. Sixty-two male Zucker rat pups shared within 15 litters received deuterated water for 5 days, prior killing at different age. Adipose tissues were collected for 2H-enrichment analyses using mass spectrometry to determine fat cell proliferation and lipid synthesis rates. Rats were assigned to obese and lean rat groups by genotyping.

Results:

 

The time course (from days 13 to 55) of all adipose tissue growth showed that the highest fractional rates of fat cell proliferation, triacylglycerol (TG) synthesis and de novo lipogenesis (DNL) took place during early suckling in all rat pups. The appearance of excessive fat mass growth in the obese rats, as compared with lean rats, was first shown through a significant increase in DNL at the end of suckling (P<0.05). The TG synthesis rate was enhanced (P<0.05) from the end of suckling and early postweaning until day 55 (from 122±10 to 498±78 in obese pups and from 25±6 to 75±26mg new TG per day in lean pups (median±s.e.m., P<0.01)). In contrast, only by day 55 did the fractional proliferation rate of fat cells in retroperitoneal and epididymal depots in the obese rats supersede that of the lean rats (P<0.05).

Conclusion:

 

The early suckling period constitutes the most active period for adipose tissue development in normal rats. In the obese Zucker rat model, adipose hypertrophy primarily contributes to the early onset of obesity, while hyperplasia increases after puberty.

Early onset of adipose tissue growth may play a determinant role in the development of obesity later in life.

Keywords:

adipogenesis, lipogenesis, fat, rat, deuterium

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Introduction

The worldwide prevalence of obesity is alarming, especially, in the increasing children population. About 10.3% American children (2–5 years) were overweight in 1999–2000, with an increase to 13.9% in 2003–2004. This increase in obesity is even more stark in male children, as prevalence increased from 9.5 to 15.1% during the same time period.1 There is growing evidence that perinatal (pre- and postnatal) environmental factors may increase the risk of developing obesity later in life.2 Accordingly, epidemiological studies suggest that small birth size and accelerated postnatal growth are also risk factors for the development of obesity.3, 4 Thus, the development of various adipose depots at an early age may be influenced by both the environment and dietary habits, and probably result in an increased susceptibility to obesity and metabolic disorders.5

Adipose tissue growth involves the formation of new adipocytes (hyperplasia) and an increase in adipocyte size (hypertrophy) by lipid accumulation. Cells in adipose tissue include mature adipocytes, in which the triacylglycerol (TG) is stored, and stromal-vascular elements such as preadipocytes, fibroblasts, endothelial cells and other vascular cells. Evaluation of the synthesis rate of TG in adipose tissue is of particular interest, since TG represents about 80% of the fat mass. Fatty acids (FAs) from adipose TG originate either from the diet or from both hepatic and adipose tissue de novo lipogenesis (DNL). Roughly, 6–12 months are required to completely replace the adipose TG stores by altering dietary FA composition in humans.6, 7 Recently, a stable isotope-mass spectrometric approach was developed for concurrent determination of lipid synthesis and cell proliferation rates in adipose tissue of rodents and humans.6, 8, 9 and 10Previous methods for measuring cell proliferation rates have fundamental limitations. They generally involve the incorporation of a labeled biosynthetic radioactive or toxic precursor, such as tritiated thymidine or bromodeoxyuridine, into cellular DNA.11 In contrast, labeling DNA using stable isotopes carries no risk and is safe. Heavy water (or deuterated water, 2H2O) and [6,6-2H2]glucose tracers have been used in vivo in humans or in rodents for determining proliferation of a variety of cell types.12, 13, 14, 15 Endogenous labeling occurs through the de novo nucleotide synthesis pathways, with detection via mass spectrometry.15 Assessing the fat cell number and size by conventional means brings an additional but static picture of the growing fat tissue.

In the present work, the ontogeny of fat tissue growth dynamics was determined by using 2H2O labeling in the lean (Fa/fa) and obese (fa/fa) Zucker rats, a model linked to leptin signaling deficiency, from the suckling period (day 13), through the postweaning period and until the young adult phase (day 55). After killing of individuals at serial time points, we measured in various fat depots the rate of incorporation of deuterium into genomic DNA, and the glycerol and palmitate moieties of TG, to determine the cell proliferation rate, total TG synthesis rate and the FA synthesis (DNL) rate, respectively.9 Our results demonstrate that the suckling period constitutes the most active period for adipose tissue development in normal rats. In obese Zucker rats, elevated lipid synthesis rates precede an increase of fat cell proliferation during the early onset of obesity.

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Materials and methods

Animals and protocol

The nonclinical experiment had a two-group parallel design. Fifteen heterozygous (Fa/fa) Zucker rat females were mated with homozygous (fa/fa) Zucker rat males (Charles Rivers, Lyon, France). Three litters were studied and killed under isoflurane anesthesia at different ages, including the suckling and postweaning periods. Three obese homozygous male pups were killed at day 13, six at day 20, seven at day 27, five at day 34 and seven at day 55. Conversely, seven lean heterozygous male pups were killed at day 13, seven at day 20, seven on day 27, seven at day 34 and six at day 55. A conventional diet (Kliba-Nafag 3434, Promivi Kliba SA, Kaiseraugst, Switzerland), which contained 18.5% protein, 54% carbohydrate, 4.5% fiber and 4.2% fat (2.03% linoleic acid, 1% oleic acid, 0.7% palmitic acid, 0.15% stearic acid and 0.02% linolenic acid), was given to all rats for the duration of the study. The source of lipids was from soybean oil and the metabolizable energy of the diet was 12.5kJg−1.

Overall, 62 male pups were utilized throughout the kinetic studies as follows.

Isotope administration procedure during suckling period
 

Five days before the killing, suckling pups of 13 and 20 days of age, and their mothers, received an intraperitoneal injection of deuterated water (35mgg−1 body weight, 99% 2H2O, 0.9% NaCl). Thereafter, the litter and mother had ad libitum access to deuterium-enriched (8–8.5% 2H2O) drinking water. The milk from the rat mothers carried deuterium-labeled water to suckling pups.

Isotope administration procedure during postweaning period
 

Pups were placed in individual cages. Five days before the killing, the individual pups received an intraperitoneal injection of deuterated water (35mgg−1 body weight, 99% 2H2O, 0.9% NaCl). They then individually had ad libitum access to deuterated (8–8.5% 2H2O) drinking water.

For both suckling and postweaning periods, blood, liver, bone marrow and mesenteric, epididymal, retroperitoneal, subcutaneous and inguinal fat tissues were sampled and weighed the day of killing. Body weight was measured throughout the 8 weeks of experimentation. The cellularity of retroperitoneal and mesenteric fat pads was also determined in the pups that were killed at day 27. The body composition was measured at day 50. A tail sample (5mm) of each rat pups was collected at killing for genotyping analyses. The ethical committee for experimentation in animals of the Swiss Authority (Canton de Vaud, nos. 1659 and 1659.1) approved the present rat study.

Analyses

Genotyping was performed by DNA analyses by Harlan UK Ltd., Hillcrest (Loughborough, UK). Body composition of pups was determined as 50 days of age using a 0.05T Magnetic Resonance Imaging (EchoMRI 400, Echo Medical Systems, Houston, TX, USA). Calculation and interpretation were performed according to Taicher et al.16 and Tinsley et al.17 Cellularity (fat cell size and number) of mesenteric and retroperitoneal fat tissues were determined through electronic quantification using the method of Hirsch and Gallian18 and improved according to Cushman and Salans 19 and Hausman et al.20 Triplicate adipose tissue samples (30–40mg) were fixed using osmium tetroxide (VWR, Geneva, Switzerland). Samples of fixed cells were analyzed on a coulter electronic particle counter (Multisizer III Beckman, Zurich, Switzerland). Counts were performed in triplicate and were reported as fraction of cells according to their size. Plasma concentrations of adiponectin (Linco, St Charles, MI, USA), insulin-like growth factor 1 (IGF-1; IDS, Tyne and Wear, UK) and insulin (Crystal Chem, Downers Grove, IL, USA) were analyzed by enzyme-linked immunosorbent assay. Enzymatic assays were used to analyze plasma concentrations of free FAs (FFAs; Wako, Neuss, Germany) and of TG (Biomérieux, Marcy l'Etoile, France).

Determination of deuterium enrichments in DNA and lipids

The measure of 2H-enrichments in DNA and in lipids has been previously described.8, 15 Briefly, cellular DNA from the adipose depot (>20mg of tissue) was isolated and hydrolyzed by sequential digestion with DNase I, nuclease P1, snake venom phosphodiesterase I and alkaline phosphatase.15, 21 An LC18 SPE column was used to separate deoxyadenosine (dA) from the other deoxyribonucleosides. Isolated dA was reduced and the deoxyribose (dR) moiety was acetylated. The resulting pentose-tetraacetate (PTA) derivative of dR was injected in ethyl acetate into the gas chromatograph/mass spectrometer (GC/MS, models 5970 or 5971, Hewlett-Packard, Palo Alto, CA, USA) for measurement of isotope enrichments. GC/MS analysis of PTA dR derivative was performed with a 30-m DB-225 column (0.25-mm i.d., 0.25-mm film thickness, J&W Scientific Folsom, CA, USA). Methane chemical ionization was used, with selected ion recording of mass-to-charge ratios (m/z) 245 and 247 (representing M0 and M2 mass isotopomers, respectively) for detection of PTA dR derivative.15

Thereafter, samples of adipose TG were isolated for analyses of 2H-enrichments of TG-glycerol and of TG-palmitate. TGs were transesterified by incubation with 3N methanolic hydrochloride. FA methyl esters were separated from glycerol by Folch's extraction. The aqueous phase containing free glycerol was lyophilized, and the glycerol converted to glycerol triacetate by incubation with acetic anhydride-pyridine, 2:1, as described elsewhere.6, 9, 22 The phase containing FA-methyl esters was concentrated under nitrogen and injected directly into the GC/MS. Glycerol-triacetate was analyzed using a DB-225 fused silica column, monitoring mass-to-charge ratios m/z 159 (parent M0) and 160 (M1), or m/z 159, 160 and 161 (M0–M1 and M2). Methane chemical ionization was used with selected ion monitoring. FA methyl esters were analyzed for composition by flame ionization detection and for 2H-enrichment by GC/MS, as described elsewhere.23 2H2O-enrichment in body water was measured in tetrabromoethane derivatized from plasma samples (15–20μl) as described previously.24 Tetrabromoethane was analyzed using a DB-225 fused silica column, monitoring m/z 265 and 266. Standard curves of known enrichments were run before and after each group of samples to calculate isotope enrichments.9

Calculations

Use of 2H2O incorporation for the measurements of TG-glycerol synthesis (all-source TG turnover), TG-palmitate synthesis (all-source DNL) and DNA replication (cell proliferation) has been described in detail elsewhere.6, 9 The calculation of cell proliferation rate is based on the precursor–product relationship.25 This method counts the cell divisions that occurred during the labeling period in the population of cells sampled by quantifying the proportion of labeled DNA strands present. The fractional proliferation rate of adipose tissue cells (fractional synthetic rate, FSRcell, in percent new cell per 5 days) identical to the fraction of newly synthesized DNA was calculated as the EM1 (DNA 2H-enrichment in percent excess) in adipose DNA divided by EM1 in bone marrow DNA.8, 21 Bone marrow enrichment was used to approximate the asymptotic or maximum DNA enrichment achievable within each animal.

The fractional and absolute TG-synthesis rate calculations have been previously described.6 The basic principle of this method is that TG synthesized from intracellular glycerol phosphate during a period of 2H2O exposure contains deuterium incorporated from tissue water. In contrast, TG that were synthesized from α-glycerol phosphate before deuterated water was present, do not contain covalent C-2H label in their glycerol moiety, thereby allowing the proportion of newly synthesized vs preexisting TG molecules to be calculated.9 The fractional synthesis rate of TG (FSRTG in percent new TG per 5 days) was calculated from the ratio of EM1TG to A1, where EM1TG is the excess mass isotopomer abundance for M1-glycerol at day 5, and A1 is the asymptotic or maximal mass isotopomer abundance for M1-glycerol, based on n=4 exchanging positions and the measured 2H-enrichment of body water (in percent excess).6 The absolute synthesis rate (in mg per day) of adipose TG was calculated from the fractional TG synthesis rate constant (kTG per day) multiplied by the adipose TG pool size.6 The fractional TG synthesis rate constant kTG (per day) was calculated from the standard precursor–product equation. The TG pool size was the adipose tissue weight (AT in mg) times 0.8 that accounts for the 80% of adipose tissue weight which is TG.

The fractional synthesis rate of palmitate (DNL contribution to palmitate, FSRpalmitate in percent new palmitate per 5 days) in TG was calculated from the label 2H-incorporation from water into TG-palmitate using mass isotopomer distribution analysis as previously described.26 The present approach measured the contribution of newly synthesized palmitate in the whole body to adipose TG synthesis (that is, liver and adipose tissue contributions are not distinguished). The present maximum 2H-enrichment reachable in palmitate was calculated based upon the measured body water enrichment, and the predicted isotopomer distribution with an N of 21 hydrogen incorporation sites.26 The absolute palmitate synthesis rate (absolute DNL in mg per day) was calculated from the fractional synthesis rate constant kpalmitate multiplied by the adipose TG-palmitate pool size.6 The fractional palmitate synthesis rate constant kpalmitate (per day) was calculated from the standard precursor–product equation. The TG-palmitate pool size was the adipose tissue weight (AT in mg) times 0.2 and 0.8 where 0.8 factor accounts for the 80% of TG in adipose tissue and 0.2 accounts for the 20% of FA which is palmitate in TG.

The fractional contribution from the DNL pathway to palmitate in newly synthesized TG, as opposed to palmitate from diet intake, was estimated as described previously,9, 10 as the ratio of FSRpalmitate to FSRTG.

Statistics

Data are presented as median±s.e.m., standard error of the median based on Rousseeuw SD(Sn). According to the distribution, two-way analysis of variance (ANOVA) with interaction (factors: age and genotype, on the data or on a transformation of the data) or Kruskal–Wallis tests (with two-way ANOVA on the rank to estimate the effect) were performed (software R2.2.1), following by appropriate contrast. A correction of Bonferroni–Sidak was applied for multiplicity of days. Day 13 was not statistically tested, because of the low number of obese pups and of a possible mother effect. Differences between tissues were assessed on the pooled data of lean and obese pups, and age independently.

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Results

Obese and lean pups

Among the 62 newborn Zucker male rat pups, 28 were homozygous and 34 were heterozygous pups. There was no difference in weight between obese (9.9±0.3g) and lean (10.0±0.2g) pups at day 3. The time course of adipose tissue weight change was progressively greater in the obese pups from day 27, as compared with lean ones, and reached significance (P<0.05) by day 55 (Table 1, Figure 1a). The liver was significantly heavier from day 27 in obese than that of lean pups (Table 1). Cells in subcutaneous and mesenteric fat tissues determined at day 27 were larger, and there were more large cells, in obese as compared to lean pups (data not shown). At day 50, the obese pups body composition comprised 36.4±0.7% fat mass and 56.4±0.5% lean mass. Conversely, the lean pups were significantly different (P<0.05), with 11.5±0.2% fat mass and 79.6±0.5% lean mass.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Determinants of the epididymal adipose tissue dynamic growth: The adipose tissue weight (a, in mg), the fractional proliferation rate (FPR) of fat cells (b, in percent new cells per 5 days), the fractional synthetic rate (FSR) of triacylglycerol (TG) (c, in percent new TG per 5 days) and DNL absolute synthesis rate of TG (d, in mg of TG per day) and the FSR of fatty acids (FA) (e, in percent new palmitate per 5 days) and the absolute synthesis rate of the FA (f, in mg of palmitate per day) measured in the epididymal adipose tissues (AT) in obese (––) and lean (––) Zucker rat pups from days 13 to 55. Data are median±s.e.m., *P<0.05, obese vs lean pups.

Full figure and legend (35K)


Fractional cell proliferation rate

2H-enrichment of genomic DNA in adipose cells ranged from 4.1±0.2 to 6.0±0.4% excess at day 13 and from 1.7±0.2 to 4.4±0.7% excess from days 20 to 55. DNA from the epididymal fat tissue cells had higher deuterium incorporation than that of other four adipose tissues. DNA 2H-enrichment in bone marrow ranged from 10.8±0.1 to 12.9±0.3% excess. The fractional proliferation rates of adipose cells of obese and lean pups are shown in Figure 2 according to their age. A significant effect of age was noticeable (P<0.05), as young pups at day 13 showed a twofold greater proliferation rate of adipose cells in all fat tissues and in both phenotypes, as compared to later in life. The subcutaneous, inguinal, mesenteric and retroperitoneal cell proliferation rates decreased considerably at day 20, and almost plateaued thereafter. At day 55, the obese pups had a significantly higher cell proliferation rate in the retroperitoneal and epididymal tissues than that of the lean pups, in which the values decreased steadily (P<0.05). The mean proliferation rate of cells in epididymal adipose tissue was higher than that in other adipose tissues. The time course development of epididymal adipose tissue cell proliferation rate is shown in Figure 1b.

Figure 2.
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The fractional proliferation rate (in percent new cells per 5 days) of the subcutaneous, inguinal, mesenteric and retroperitoneal adipose tissue (AT) cells in obese (––) and lean (––) Zucker rat pups from days 13 to 55. Data are median±s.e.m., *P<0.05, obese vs lean pups.

Full figure and legend (26K)

TG-synthesis rate

2H-enrichments of TG-glycerol in adipose tissues ranged from 2.5±0.6 to 12.7±0.4% excess. 2H-enrichment of body water ranged from 3.8±0.1 to 5.4±0.1% excess. The fractional synthesis rate of TG according to age in epididymal adipose tissue of obese and lean pups is shown in Figure 1c. A significant age effect was evident (P<0.05), as pups at day 27 showed a three- to fourfold decrease in fractional TG synthesis rate, as compared with day 13 in both groups. In all tissues, fractional TG synthesis rate nearly plateaued from day 34. However a small, but significant, increase was observed in the obese pups at day 34 in the five fat tissues as compared with the lean pups (P<0.05, except mesenteric depot). The fractional TG synthesis rate in obese pups diminished thereafter down to values measured in lean pups at day 55. The mean fractional TG synthesis rate was similar in epididymal and mesenteric fat pads. These were higher than those in subcutaneous, inguinal and retroperitoneal fat pads (data not shown).

The absolute synthesis rate of TG in fat tissues of obese and lean pups is shown in Figures 1d and 3. A significant effect of age was observed in the obese pups (P<0.05). From day 27, the absolute rate was markedly elevated in all fat tissues in obese pups as compared with lean pups (P<0.05, only from day 34 for mesenteric depot). The mean absolute TG synthesis rate was higher in subcutaneous than those in other tissues.

Figure 3.
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The absolute synthesis rate of triacylglycerol (TG) (in mg of TG per day) in the subcutaneous, inguinal, mesenteric and retroperitoneal adipose tissues (AT) in obese (––) and lean (––) Zucker rat pups from days 13 to 55. Data are median±s.e.m., *P<0.05, obese vs lean pups.

Full figure and legend (25K)

De novo lipogenesis

2H-enrichments of TG-palmitate in adipose tissues ranged from 3.5±0.7 to 19.1±0.9% excess. The fractional synthesis rate of palmitate in epididymal adipose TG of obese and lean pups is shown in Figure 1e. A significant effect of age was noticeable (P<0.05), as pups at day 20 showed a twofold decrease as compared with younger aged lean pups. The fractional DNL synthesis rates in subcutaneous, inguinal and retroperitoneal tissues plateaued from day 34 in lean pups. The mesenteric and epididymal fractional DNL synthesis rates showed a smooth decrease from days 13 to 55 in lean pups (Figure 1e). The fractional DNL contribution to palmitate evolved differently during the growth of obese pups. While the DNL value decreased with age in lean pups, a remarkable increase occurred in obese pups at the age of 27–34 days, with a peak value at day 34. Thereafter, the fractional DNL rate decreased in the 55-day-obese pups and reached values close to fractional values in the lean pups (Figure 1e).

Although this method does not distinguish hepatic from adipose-derived DNL, differences appeared among measurements in the fat tissues. The fractional DNL measured in epididymal fat tissue was higher than that in all other fat tissues. Fractional DNL measured in mesenteric fat tissue was higher than that in inguinal and retroperitoneal fat tissues, which were higher than those in subcutaneous fat tissue (data not shown).

The absolute synthesis rates of palmitate (absolute DNL) measured in fat tissues of obese and lean pups are shown in Figures 1f and 4. A significant effect of age was apparent, as the absolute DNL increased dramatically from days 20 to 34 in all fat tissues (P<0.05). From day 34, either the absolute DNL plateaued when measured in the subcutaneous and mesenteric tissues, or the increase was less marked in other fat tissues in obese pups. The absolute DNL was higher when measured in the subcutaneous tissues as compared to other tissues.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The absolute synthesis rate of fatty acids (absolute de novo lipogenesis (DNL), in mg of palmitate per day) measured in the subcutaneous, inguinal, mesenteric and retroperitoneal adipose tissues (AT) of obese (––) and lean (––) Zucker rat pups from days 13 to 55. Data are median±s.e.m., *P<0.05, obese vs lean pups.

Full figure and legend (24K)

DNL contribution to new adipose TG synthesis

The DNL contribution to newly synthesized TG was calculated and is shown in Table 2. Higher ratios of FSRPalmitate/FSRTG than 100% were observed in obese pups at days 27 and/or 34, consistent with partial recycling of mono- and diglycerides from hydrolysis to resynthesis of TG in adipose tissue cells.


Plasma biomarkers

A significant effect of age was observed in the plasma concentrations of adiponectin, IGF-1, insulin and TG (P<0.05), while no effect of age was observed in the concentration of FFA. The adiponectin concentrations were significantly higher in obese than those in lean rats (Table 3), with a ratio of adiponectin-to-fat mass being higher in lean (0.49±0.03) than that in obese rats (0.23±0.01 (μgml−1)/g of body fat, P<0.05) at day 55. No difference was shown at different ages in IGF-1 (Table 3). The insulin and TG concentrations were higher in obese than those in lean pups after day 27 (P<0.05, Table 3). A higher FFA concentration was shown in obese than that in lean pups only at day 55 (P<0.05, Table 3).


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Discussion

Adipose tissue growth involves both hyperplasia and hypertrophy. In the present study, we used deuterated-water intake to determine adipogenic and lipogenic components of adipose tissue growth in young lean and obese Zucker pups. The 2H-enrichment in body water reached a value of ~5%, that is consistent with the previous study of Antelo and co-workers,8, 15, 27 who observed a plateau of about 2.5% when rats were given half the dose of the present study for 11 weeks.14 According to the previous work, the present approach allows an assessment of 2H-enrichment in genomic DNA and lipids of fat cells from sample biopsies of as little as 10–20mg of fat tissue.

During adipose tissue development from suckling to puberty, the phase of highest fat cell hyperplasia has been illustrated to take place from birth up to day 30 in normal rats.28 In the current study, lean heterozygous (Fa/fa) Zucker rats demonstrated the most active adipose fractional cell proliferation rate during the suckling period (days 8–13 after birth; the earliest measurable interval). However, the fractional cell proliferation rate sharply decreased at the end of the suckling period (from days 15 to 20) and remained nearly constant throughout puberty (day 55) in subcutaneous, inguinal and retroperitoneal fat pad, and continued to slowly decline in mesenteric and epididymal adipose tissues. These data indicate that in this model, a fast growing rate of adipose tissue cells takes place during the middle part of (and probably before) the suckling period. Because of limitations in fat tissue availability, the separate measurement of the proliferation rate in the adipocyte vs the stromal vascular fraction was not feasible in our study. Therefore, it is not possible to estimate the rate of newly divided mature adipocytes. By using 3H-labeling, Greenwood et al.29 showed an active synthesis of new adipocytes in epididymal fat pad of 9-day-rat pups, while some adipose cell proliferation continue to occur during the postweaning and pre-pubertal period but stopped after puberty. At puberty we observed limited adipose tissue cell proliferation rate (3–4% new cells produced per day) in the different fat depots of the heterozygous lean Zucker rats. By using the same deuterium labeling methodology, Antelo and co-workers8, 14 observed a proliferation rate of mature adipocyte-enriched cells of about 0.4–0.7% and 1–1.5% new cells produced per day in adult rats and mice, respectively.

Adipose TG synthesis and DNL rates have been previously measured in adult rodents by using 2H2O labeling techniques.6, 27 Nevertheless, to our knowledge, this is the first time that adipose lipid synthesis rate is assessed in vivo during early life. The issue of cell hypertrophy was addressed by comparing lipid dynamics to cell dynamics in adipose tissue depots. Fractional TG turnover and fractional DNL rates were highest during the first phase of the suckling period. In rat, body fat content at birth is very low (1% of body weight) and white adipose tissue is barely detectable.28 At day 13, the sum of the different fat pad weights of the lean Zucker rats was already about 0.8g, indicating an intense adipose tissue growth during the early postnatal life. The absolute TG and DNL synthesis rates in most fat pads were quite constant during the postweaning period, but increased steadily around puberty, probably due to hormonal influences. This lipid synthesis profile was similar to that of the adipose tissue weight gain, with a constant positive slope until day 55 at puberty in the lean rat pups. Hepatic and adipose lipogenic enzymes have been previously shown to increase during the suckling–weaning transition in all rats.30, 31 In the present study, we did not observe a marked elevation of the fractional or absolute DNL rate during this transitory period in normal rats. Nevertheless, the contribution of DNL (vs the diet) to the pool of TG-palmitate was higher during the postweaning than that of the suckling period, for subcutaneous, inguinal and retroperitoneal adipose tissues. Because our measurements include both adipose and hepatic DNL, the observed difference in DNL measured in the various fat tissues could originate either from a true difference in the tissue's lipogenic activity, or from a different lipoprotein lipase (LPL) activity at the fat tissue membrane.32, 33 Another factor that could influence fat accrual is the breakdown rate, which we did not measure. Brunengraber et al.27 have previously shown that adipose TG degradation occurs at about 50% of the rate of synthesis in adult rats. Overall, these results indicate that both cell proliferation and lipid synthesis contribute to the intense adipose tissue development occurring during the early postnatal period in normal rats. Therefore, the suckling period appears to be an important window for potential programming of adipose tissue by environmental factors such as nutrition.

The comparison of adipose tissue growth dynamics during early life between lean and genetically obese rats revealed interesting findings. Until puberty, the highest adipose tissue growth observed in the obese Zucker rats, as compared with the heterozygous lean phenotype, is not explained by an increase in adipose cell hyperplasia. Indeed, the fractional cell proliferation rate was not significantly different between lean and obese rats from days 13 to 34, but was increased in subcutaneous, retroperitoneal and epididymal fat depots of 55-day-obese rats. This result is in agreement with previous data obtained by the classical method of cell counting. When compared with the lean Zucker rat, the number of adipocytes per inguinal fat pad was not increased in the obese phenotype between the age of 7–28 days, but was twofold higher at day 56.34 While cell proliferation did not appear to contribute to the fat mass excess of the obese rats in early life, the increase of lipid synthesis was revealed to be a major contributor. At the end of the suckling period, the fat pad weight, the absolute TG synthesis and DNL rate were higher in the subcutaneous, inguinal and retroperitoneal fat pads of the obese pups, as compared with the lean phenotype. This hypertrophic phase is probably not linked to hyperphagia, since it has been previously shown that this phenomena initiates during the weaning period, and that the obese pups do not ingest more milk than that of lean animals when suckling.35, 36 Furthermore, diet was not contributing more to TG synthesis in obese pups than that in lean pups during the suckling period (as observed from FSRPalmitate/FSRTG ratio; Table 2). In addition, it has been clearly established that hyperphagia is not required in the development of obesity in the Zucker obese rats.37, 38 The active hypertrophy at the end of suckling may originate from (1) an increase in lipogenic-enzyme activity, (2) more GLUT-4 glucose-transporters on the adipocyte membrane or/and (3) a more efficient LPL transport of FA into the cells. In the present study, the elevated insulin that is initiated at the end of suckling in obese pups probably favored lipogenic and GLUT-4 activities. Pénicaud et al.31 showed increased mRNA levels and activities of lipogenic enzymes (ACC and FAS) and of GLUT-4 at the end of suckling especially when switching from milk to a high-carbohydrate diet. Interestingly, Dugail et al.32 observed that LPL activity is already enhanced in 7-day-old obese Zucker rat pups as compared to lean counterparts before the end of suckling.

A further dramatic increase of both absolute TG synthesis and DNL was documented 2 weeks after weaning in the obese rats as compared with the lean rats. This phenomenon was linked to a significant increase in the fractional TG synthesis and DNL rates, and could be due to the onset of hyperphagia and activation of FA synthetase at this age, respectively. The increased DNL contributed predominantly (as opposed to FAs from the diet) to the increased synthesis of TG 2 weeks after weaning in obese pups. Interestingly, the fractional DNL rate sharply decreased (and the absolute DNL reached a plateau) around puberty, and for most adipose tissues was not significantly different than what observed in the lean animals. During this period, the fractional cell proliferation rate of different adipose tissue depots started to be significantly greater in the obese rats as compared with the lean ones. From our observation, neither IGF-1 nor adiponectin concentrations were associated with the fat cell proliferation rate. The present in vivo study did not support previous in vitro studies that identified IGF-1,39 as well as adiponectin,40 as fat cell proliferation stimulators. The evolution of insulin concentration along the age was associated with the one of fat cell proliferation in the obese phenotype. But to our knowledge insulin has not been shown to play a direct role in hyperplasia.41 Further investigation would be needed to better understand the switch from a hypertrophy to a hyperplasia active phase.

Finally, the use of deuterated water to label genomic DNA and lipids is pertinent to determine the fractional proliferation rates of fat tissue cells, as well as to measure simultaneously the TG synthesis rate and DNL in rat pups. In conclusion, an excess of adiposity starts to take place in different fat depots during the late phase of the suckling period in the obese Zucker rat model. Up to 2 weeks after weaning this excess of adiposity is due to an increase in TG synthesis, in parallel to an enhanced DNL contribution, but not to fat cell proliferation. Five weeks after weaning, both hyperplasia and hypertrophy in certain fat depots contribute to the obesity phenotype.

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Acknowledgements

We particularly thank Maryse Barbier, Corinne Ammon-Zufferey, Irina Monnard, Ornella Avanti-Nigro, Iréne Zbinden, Florence Blancher and Julie Moulin for their constructive advice and collaboration in the animal study. We are grateful to Trent Stellingwerff. We also thank the animal house staff, especially Massimo Marchesini, Laurent Potier and Christophe Maubert. The present work was financed by Nestec Ltd. and has been carried out in the frame of the European Community project entitled ‘Early nutrition programming-long term follow up of efficacy and safety trials and integrated epidemiological, genetic, animal, consumer and economic research’, EARNEST (Food-CT-2005-007036 FP 6 priority 5.4.3.1 Food quality and safety).

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