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Keywords:

  • food intake;
  • leptin;
  • adipose tissue

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: To characterize the effect of feeding conditions on the expression behavior of adiponutrin, a non-secreted adipose-specific protein proposed to be involved in energy homeostasis maintenance, and its relation with leptin expression in different rat adipose tissue depots under normal and obese conditions.

Research Methods and Procedures: Two rat models were used, Wistar (lean and overweight) and Zucker (lean and obese), submitted to fasting/refeeding. Adiponutrin and leptin expression was determined in different white adipose tissue depots (epididymal, inguinal, mesenteric, and retroperitoneal) and in interscapular brown adipose tissue by reverse transcription-polymerase chain reaction.

Results: We have found site-specific differences in adiponutrin expression in different adipose depots, being the expression pattern close to that of leptin in white adipose tissue. The depot-specific adiponutrin expression is similar in lean and obese animals, except in the inguinal depot, where adiponutrin is overexpressed in obese Zucker. Independently of the degree of expression in the tissue, adiponutrin is an acute sensor of feeding conditions compared with other diet-regulated adipokines, as is leptin. In lean rats, 14-hour fasting greatly decreases adiponutrin mRNA levels in all of the depots studied, whereas 3-hour refeeding allows the recovery of the levels found in control animals. In both overweight Wistar and obese Zucker rats, the decreased mRNA expression observed after fasting in lean rats is not as evident; moreover, in the obese Zucker, there is no recovery after refeeding.

Discussion: Adiponutrin expression is highly regulated by feeding conditions in the different adipose tissue depots, but this regulation is impaired in obese rats.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Adiponutrin is a non-secreted transmembrane protein of 413 amino acid residues that is expressed mainly in adipose tissue (1). Adiponutrin expression is markedly increased during in vitro 3T3-L1 preadipocyte differentiation (1, 2) and is under tight nutritional regulation, dramatically decreasing after fasting and being strongly up-regulated by feeding (1, 3, 4, 5). Diet macronutrient composition has also been found to affect adiponutrin expression, that is, refeeding with a high-sucrose or a high-protein diet produces a rapid up-regulation (3, 4), whereas a high-fat diet produces no change (4). Nutritional regulation of adiponutrin seems to be mediated by insulin and glucose because adiponutrin expression in vitro and in vivo is up-regulated in response to both signals (1, 6, 7). In addition, it has been described that adiponutrin is highly expressed in obese Zucker rats (1). For these reasons, adiponutrin has been proposed to be involved in energy homeostasis and adipocyte function (1, 5). Recently, two human adiponutrin polymorphisms have been demonstrated to be associated with obesity (7).

It has been shown that adiponutrin belongs to the phospholipase A2 family, being described as iPLA2ε (2). This protein has a robust triacylglycerol lipase activity and, moreover, has acylglycerol transacylase activity using monoolein as an acyl donor to a diolein acceptor to produce triacylglycerols, thus representing a previously unrecognized acyl-CoA-independent pathway for triacylglycerol biosynthesis (2). Hence, adiponutrin could participate in lipid homeostasis by facilitating energy mobilization and storage in adipocytes (2). However, it has been observed that small interfering RNA-mediated knockdown of adiponutrin in 3T3-L1 adipocytes has no effect on glycerol or non-esterified fatty acid release (6) and that overexpression of adiponutrin has no effect on intracellular triacylglycerol levels (8); thus, its exact biological function still remains unclear. It has been proposed that adiponutrin could be part of a triacylglycerol recycling cycle in the adipocyte, a futile cycle that could enable the rapid response to changing metabolic conditions such as fasting/refeeding (2).

Adiponutrin shares many of the characteristic features of adipose-specific adipokines regarding regulation by food intake, although it does not seem to be secreted and so it cannot be exactly defined as an adipokine. Adiponutrin expression has been related to the same regulatory pathways as leptin, although the exact relation between these two proteins has not yet been established. In fact, adiponutrin has been described to be more closely related to leptin than to any other adipose-derived protein with respect to the acute regulation of its expression by meal feeding (3).

With the present knowledge, we can say that adiponutrin acts as an acute sensor of the energy state regulated by diet, rapidly responding to feeding and fasting. Nutritional regulation of adiponutrin in animals with a normal body weight has already been described, but there are no data regarding the response of adiponutrin expression to fasting/refeeding conditions in obese animals, and only few data exist describing site-specific differences in adipose expression. In the present study, the regulation of adiponutrin by feeding conditions (fasting and refeeding) in different adipose tissue depots, both in normal and obese rats, has been studied. We have also studied the relation between diet regulation of adiponutrin expression and diet regulation of leptin in different rat adipose depots.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Three-month-old male Wistar rats (Charles River Laboratories España SA, Barcelona, Spain) and 3-month-old male Zucker rats, both lean (_/?) and obese (fa/fa) (Charles River Laboratories España SA) fed with a standard chow diet (Panlab, Barcelona, Spain) were used. Rats were distributed into three groups (n = 5): a control-fed group, animals provided with ad libitum access to chow diet; a fasted group, animals deprived of food for 14 hours; and a refed group, fasted animals with a posterior free access to chow diet for 3 hours. An extra group of Wistar rats (n = 5) fed for 6 months after weaning (6.7 months old) with a hyperlipidic diet (with 45% of calories from fat) (Research Diets, Inc., New Brunswick, NJ) was used to understand whether the results obtained for feeding regulation of adiponutrin in obese Zucker rats were due to the lack of leptin receptor or to other factors related to the excess of body weight. In this case, hyperlipidic diet was used instead of chow diet for the refed group. A group of 6.7-month-old lean-fed Wistar rats was used as age control for the overweight Wistar. All of the animals were housed at 22 °C with a period of light/dark of 12 hours and with free access to food and water.

After killing the animals, different white adipose tissue depots [epididymal white adipose tissue (EWAT),1 inguinal white adipose tissue (IWAT), mesenteric white adipose tissue (MWAT), and retroperitoneal white adipose tissue (RWAT)] and the interscapular brown adipose tissue (IBAT) were rapidly removed, weighed, and frozen in liquid nitrogen and stored at −70 °C until RNA analysis. Blood was also collected, stored at room temperature for 1 hour and overnight at 4 °C, and was then centrifuged at 1000g for 10 minutes to collect the serum. Guidelines for the use and care of laboratory animals of our University were followed.

Adiposity Index

Adiposity was determined by an adiposity index computed for each rat as the sum of EWAT, IWAT, MWAT, and RWAT depot weights and expressed as a percentage of total body weight.

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Analysis of Adiponutrin and Leptin mRNA

Adiponutrin and leptin expression was determined in different white adipose tissue depots (EWAT, IWAT, MWAT, and RWAT) and in IBAT by RT-PCR. Total RNA was extracted using Tripure reagent (Roche, Barcelona, Spain) according to the instructions provided by the supplier. Total RNA (0.5 μg; in a final volume of 10 μL) was denatured at 90 °C for 1 minute and then reverse transcribed to cDNA using murine leukemia virus reverse transcriptase (according to procedure of Applied Biosystems) at 42 °C for 1 hour, with a final step of 5 minutes at 99 °C in a Perkin Elmer 9700 Thermal Cycler (Norwalk, CT). Half of the RT product was used for adiponutrin/leptin amplification and the other half for β-actin amplification using the AmpliTaq Gold DNA polymerase (Applied Biosystems, Madrid, Spain). Samples were first denatured at 94 °C for 3 minutes, and then PCR was carried out using the following parameters: 94 °C for 1 minute, 58 °C for 1 minute, and 72 °C for 2 minutes. For adiponutrin, the number of cycles was 22 for lean Wistar and for lean and obese Zucker rats and 27 cycles for overweight Wistar rats. A comparative PCR for all Wistar and Zucker rats was carried out at 22 cycles to have the possibility to compare all of the samples. For leptin and β-actin, the number of cycles was 23 and 22, respectively, for all of the animal models. The amplification was finished by a final extension step of 10 minutes at 72 °C. Primers for the adiponutrin gene were as follows: f5′-GTTTGCAGGCTGCGGCTTCC-3′ and r5′-GGCAGATGTCATGCTCACCG-3′; for the leptin gene, f5′-CCAGGATGACACCAAAACCCTC-3′ and r5′ATCCAGGCTCTCTGGCTTCTGC-3′; and for the β-actin gene, f5′-GAAGCTGTGCTATGTTGCCC-3′ and r5′-GGATTCCATACCCAGGAAGG-3′. The expected size of the products was 550 bp for the adiponutrin gene, 316 bp for the leptin gene, and 164 bp for the β-actin gene, which were visualized by electrophoresis in a 1.5% agarose gel containing ethidium bromide and verified by using a DNA 100-bp ladder. The bands in the gel were quantified in a Chemigenius BioImaging System (Syngene, Cambridge, UK), using the GeneTools Software (Syngene). The signals for adiponutrin and leptin mRNA were normalized to the signal of the housekeeping gene β-actin, and the results were expressed as the adiponutrin or leptin-to-β-actin mRNA ratio. The housekeeping gene expression did not change depending on the different adipose tissue depots nor on feeding conditions nor on the lean/obese-overweight state for any of the studied adipose depots (one-way ANOVA, p < 0.05).

Quantification of Insulin and Leptin Levels

Insulin concentration in serum was measured with a rat insulin enzyme-linked immunosorbent assay (ELISA) kit (DRG Instruments GmbH, Marburg, Germany) following standard procedures, and leptin was measured with a mouse ELISA kit (R&D Systems, Minneapolis, MN).

Statistical Analysis

All data are expressed as the mean ± standard error. The statistical significance of differences in body weight, adiposity index, adiponutrin and leptin expression, and serum insulin and leptin in the different adipose tissue depots for the different feeding conditions and in the different rat models was assessed by one- and two-way ANOVA and least significant difference post hoc comparisons. Linear relationships between key variables were tested using Pearson's correlation coefficients. The analyses were performed with SPSS for Windows (SPSS, Chicago, IL). Threshold of significance was defined at p < 0.05 and is indicated when different.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Body Weight and Serum Parameters of the Animals

Lean Wistar and lean and obese Zucker rats were of the same age (3 months old) and overweight Wistar rats were older (6.7 months old). As shown in Table 1, obese Zucker rats attained 18% higher body weight in comparison with lean Zucker animals, whereas Wistar rats fed with a hyperlipidic diet attained 11% overweight in comparison with lean Wistar rats of the same age. Both obese/overweight models were insulin resistant and hyperleptinemic; in Zucker rats, insulin and leptin levels were, respectively, 25.7- and 11.9-fold higher in obese vs. lean animals, and in Wistar rats, levels were 3.6- and 1.9-fold higher in overweight vs. lean Wistar rats of the same age.

Table 1.  Body weight, adiposity index, and serum parameters (insulin and leptin) of the different animal models used
 WistarZucker 
  • The adiposity index was computed as the sum of epididymal, inguinal, mesenteric, and retroperitoneal white adipose tissue depot weights and expressed as a percentage of total body weight. Insulin and leptin levels were measured by enzyme-linked immunosorbent assay. Results represent mean ± standard error (n = 5).

  • *

    Overweight/obese vs. lean animals of the same age (one-way ANOVA, p < 0.05). O, effect of body weight; M, effect of the animal model; OxM, interaction of body weight and animal model (two-way ANOVA, p < 0.05), considering data of 6.7-month-old Wistar (lean and overweight) and Zucker (lean and obese) rats.

 Lean (3 months)Lean (6.7 months)Overweight (6.7 months)Lean (3 months)Obese (3 months) 
Body weight (g)346 ± 12553 ± 15616 ± 17*331 ± 5403 ± 9*O, M
Adiposity index (%)4.25 ± 0.308.16 ± 0.4412.50 ± 1.28*2.52 ± 0.228.69 ± 0.13*O, M
Insulin (ng/mL)0.12 ± 0.020.69 ± 0.282.46 ± 0.25*0.57 ± 0.1314.67 ± 3.71*O, M, OxM
Leptin (pg/mL)4591 ± 5098845 ± 146116953 ± 2061*3465 ± 23941080 ± 2900*O, M, OxM

Correlation of Adiponutrin and Leptin Expression with Body Weight, Adiposity, and Serum Insulin and Leptin Levels

Adiponutrin and leptin expression correlated positively with body weight (R = 0.851, p < 0.01 and R = 0.936, p < 0.01, respectively), adiposity index (R = 0.798, p < 0.01; R = 0.956, p < 0.01), serum insulin (R = 0.684, p < 0.05; R = 0.844, p < 0.01), and serum leptin (R = 0.775, p < 0.01; R = 0.901, p < 0.01) when considering Zucker animals (both lean and obese) but only in the IWAT depot, whereas no correlation was found for any of the other depots or when considering the data of all of the depots together. Again, it was in the IWAT depot where adiponutrin and leptin expression correlated positively (R = 0.903, p < 0.01), but a correlation considering all of the studied adipose depots together also was found for the expression of these two genes (R = 0.334, p < 0.05). The expected correlations of serum leptin with body weight, adiposity index, and serum insulin were also found (data not shown).

Adipose Tissue Depot-Specific Differences in Adiponutrin and Leptin Expression

Adiponutrin mRNA levels varied depending on the adipose tissue depots (one-way ANOVA, p < 0.05) (Figure 1). In Wistar rats fed with a standard chow diet (lean rats) and in non-obese Zucker rats, the pattern of adiponutrin expression was similar. The highest mRNA levels were in the brown adipose tissue and in the EWAT and RWAT depots, followed by the MWAT one, whereas the IWAT depot showed only a slight adiponutrin expression (Figure 1A and 1C). It is noticeable that in obese Zucker rats, the pattern was essentially as commented above, although adiponutrin expression was markedly increased in the IWAT depot (Figures 1C and 2B). In Wistar rats fed for 6 months with a hyperlipidic diet (diet-induced overweight rats), the expression pattern was different, with a higher expression in the brown adipose tissue than in the different white adipose depots, being here again outstanding that the expression was increased in the IWAT depot (p = 0.06, Student's t test) in comparison with lean Wistar (Figures 1B and 2B).

image

Figure 1. Adiponutrin mRNA expression levels in different white adipose tissue depots (EWAT, IWAT, MWAT, RWAT) and in IBAT in different feeding conditions in different animal models: 3-month-old Wistar rats (A), Wistar rats fed a high-fat diet for 6 months after weaning (6.7 months old) (B), and 3-month-old lean and obese Zucker rats (C), measured by RT-PCR. Fed animals had ad libitum access to food, fasted animals were deprived of food for 14 hours, and re-fed animals were given free access to food for 3 hours after 14 hours of fasting. Results are comparable between the animal groups and represent mean ± standard error (n = 5) of ratios of specific mRNA levels to β-actin [arbitrary unit(s) (Au)]. T, effect of the adipose tissue depot; F, effect of feeding conditions; O, effect of body weight; T × F, interaction of adipose depot and feeding conditions; F × O, interaction of feeding conditions and body weight (two-way ANOVA, p < 0.05). Within each animal model, bars not sharing a common letter (a to c) are significantly different (one-way ANOVA, p < 0.05). * vs. control-fed rats (one-way ANOVA, p < 0.05). # IWAT of obese Zucker rats vs. IWAT of lean Zucker rats (p < 0.05, Student's t test).

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image

Figure 2. Comparison of adiponutrin expression levels in different rat models [3-month-old Wistar rats, lean Wistar rats; 3-month-old lean and obese Zucker and Wistar rats fed a high-fat diet for 6 months after weaning (6.7 months old), overweight Wistar rats] in the RWAT (A) and IWAT (B) adipose tissue depots, measured by RT-PCR. A representative RT-PCR is shown. β-Actin was used as housekeeping gene. In the graph, results represent mean ± standard error (n = 5) of ratios of specific mRNA levels to β-actin [arbitrary unit(s) (Au)]. M, effect of the animal model; O, effect of body weight; M × O, interaction of animal model and body weight (two-way ANOVA, p < 0.05). Bars not sharing a common letter (x to z) are significantly different (one-way ANOVA, p < 0.05).

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Leptin expression, as already known (9, 10), also had site-specific differences (one-way ANOVA, p < 0.05) (Figure 3). Wistar lean rats had a low expression in brown adipose tissue and in the IWAT, followed by the MWAT depot, with the highest expression levels in the EWAT and RWAT depots (Figure 3A); the pattern of expression was very similar in lean Zucker rats (Figure 3C). In overweight Wistar and obese Zucker rats, leptin expression was increased in the brown adipose tissue and in the IWAT depot (Figure 3B and 3C).

image

Figure 3. Leptin mRNA expression in the same feeding conditions, tissues, and animal models described in Figure 1.

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Regulation of Adiponutrin and Leptin Expression by Feeding Conditions in Different Adipose Tissue Depots

Feeding conditions affected adiponutrin expression both in lean animals and in animals with an excess of body weight (one-way ANOVA, p < 0.05) (Figure 1). In lean rats, independently of the specific degree of adiponutrin expression in the tissue, 14-hour fasting highly decreased mRNA levels in all of the adipose depots of Wistar rats (approximately 85%) and Zucker rats (approximately 75%), whereas 3-hour refeeding allowed the recovery of the levels found in the control animals (Figure 1A and 1C). Nevertheless, in overweight Wistar and in obese Zucker rats, fasting produced a decrease that was not as evident as in the other models (Figure 1B and 1C). In Wistar rats adiponutrin significantly decreased with fasting in all of the studied depots (approximately 48%), except in the EWAT and in MWAT, where the decrease was less pronounced (12% and 32%, respectively), and in Zucker rats, the decrease was significant in all of the white adipose depots (approximately 58%) but not in the brown adipose tissue (40%). Moreover, in these rats, the adiponutrin levels found in control-fed animals were recovered in Wistar but not in Zucker rats.

Regarding leptin nutritional regulation, as expected (1, 10), its expression was affected by feeding conditions in lean Wistar and lean Zucker rats (one-way ANOVA, p < 0.05) (Figure 3A and 3C), in the same manner as adiponutrin, decreased expression by fasting and recovery of the initial fed levels with refeeding. However, leptin decrease with fasting in the different adipose depots (approximately 50% in Wistar and 41% in Zucker rats) was not as marked as for adiponutrin. Also, as expected (10), fasting/refeeding did not affect leptin expression levels in obese Zucker rats (Figure 3C).

Comparison of Adiponutrin Expression Levels between the Different Rat Models

Adiponutrin mRNA levels were differently expressed when comparing the same adipose tissue depots between the different rat models (one-way ANOVA, p < 0.05). Wistar rats tended to express higher adiponutrin mRNA levels in the different adipose depots than Zucker rats, both lean and obese (to see the RWAT as a representative depot, see Figure 2A), with the exception of the IWAT depot, where adiponutrin was overexpressed in obese Zucker and increased in overweight Wistar (Figure 2B) rats. In Wistar rats fed a hyperlipidic diet for 6 months, adiponutrin levels were highly reduced in all of the studied depots in comparison with the other models.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Depending on its anatomical localization, the adipose organ exhibits strong heterogeneity in terms of physiological and biochemical properties (11), which could indicate different regulatory mechanisms in the different adipose depots. In fact, there are important differences in adipokine expression and regulation depending on the adipose depot (9, 12, 13, 14). Moreover, the obese state is associated with alterations in adipokines production and regulation in comparison with the lean state (10, 15, 16). Here, we describe that the expression of adiponutrin, a recently identified protein mainly expressed by the adipose tissue and regulated by feeding conditions, has site-specific differences in the different adipose tissue depots and is regulated by fasting/refeeding in a different manner depending on the rat model used and on the body weight (normal or obese conditions).

There are only very few data available on adiponutrin expression in different adipose tissue depots. In rats, a higher expression in the EWAT depot has been described in comparison with the subcutaneous adipose depot (5), and in the first work describing adiponutrin, a high expression was also found in the brown adipose tissue of mice (1). However, no other specific work has been published up to now. Our data indicate that there are site-specific differences in adiponutrin expression in rats, with a higher expression in the brown adipose tissue and in the EWAT and RWAT depots, followed by the MWAT one, and with the lowest adiponutrin mRNA levels in the IWAT depot, which is subcutaneous fat, with a similar expression pattern in lean Wistar and Zucker rats.

Adiponutrin expression has been related to the same regulatory pathways as leptin more than to any other adipose-specific product (3, 5). Interestingly, the differential expression pattern found here for adiponutrin in the different white adipose tissue depots is the same as the one previously described for leptin (9), which could also indicate similarities in the regulation of both proteins.

It is known that the expression of different adipokines, such as leptin, is altered in different obese and insulin-resistant models (16, 17), so adiponutrin could also be affected. Baulande et al. (1) have reported that adiponutrin is up-regulated in obese Zucker rats; based on this fact, a link between adiponutrin and obesity has been postulated. These authors found that adiponutrin mRNA levels were 50-fold elevated in the brown adipose tissue and in the IWAT depot of genetically obese Zucker rats compared with lean ones (1). Our data also show that adiponutrin expression is increased in obese Zucker rats, but this is only evident in the IWAT depot, whereas in the other white adipose tissue depots, the levels of expression are similar in obese and non-obese animals. It is of note that the up-regulation of adiponutrin is observed precisely in the depot that has the lowest expression in lean animals. In fact, as a result of the obese condition, the IWAT depot begins to express adiponutrin mRNA levels comparable with those of the other adipose depots, thus suggesting that this overexpression could be part of a metabolic adaptation in response to the increase in body weight. In fact, adiponutrin only correlated positively with body weight and adiposity index when considering this depot. Regarding brown adipose tissue, our data do not demonstrate an overexpression when comparing lean and obese animals as described by Baulande et al. A reason for this difference could be the existence of gender-associated differences in adiponutrin expression because Baulande et al. used female rats, whereas our animals were male. The existence of sexual differences in brown adipose tissue protein expression and regulation has been observed before, for example, for uncoupling protein-1, the specific protein of this tissue responsible for thermogenesis (18, 19). In humans, no differences had been reported in adiponutrin mRNA levels in subcutaneous adipose tissue of lean and obese women (20), but it has been described recently that obese subjects have increased adiponutrin mRNA levels in subcutaneous and visceral abdominal adipose tissue (7).

Adiponutrin expression is under a strong nutritional regulation that has been previously characterized in animals with normal body weight; its mRNA levels greatly decrease with fasting and are restored with refeeding (1, 3, 4, 5). Nevertheless, as far as we know, there are no previous data regarding the response of adiponutrin expression to fasting/refeeding conditions in obese animals, and our results show that there is a different adiponutrin regulation by feeding in the obese state.

Adiponutrin expressed in the two different strains of lean rats studied, Wistar and Zucker, has a similar feeding regulation pattern, coincident with that previously described by other groups (1, 3, 4, 5), although it is remarkable here that our fasting conditions were shorter in time (14 hours) than the ones previously tested (19 and 21 hours). Independently of the specific degree of expression in the tissue, 14-hour fasting is enough to greatly decrease adiponutrin mRNA levels in all of the studied adipose depots. The 3 hours of refeeding allowed the recovery of the levels found in the control animals, in both rat strains, Wistar and Zucker. As expected, leptin regulation by fasting/refeeding followed a similar pattern to adiponutrin (1, 21). Fasting decreased leptin mRNA levels in the different adipose depots, whereas refeeding allowed the recovery of the levels of control-fed animals. However, comparing both proteins, leptin decrease with fasting was less marked.

When studying obese animals, adiponutrin response to fasting/feeding was found to be altered. In response to fasting, the decrease in mRNA levels was not as evident in obese Zucker as in lean animals; moreover, levels remained low after refeeding. Genetically obese Zucker rats lack the functional leptin receptor, which determines the development of obesity accompanied by hyperinsulinemia and insulin resistance (22, 23, 24). Both facts, the lack of a functional leptin receptor and/or hyperinsulinemia, could affect adiponutrin expression. In fact, insulin has been shown to up-regulate adiponutrin expression in vitro, and the effect is glucose-dependent (1, 6, 7). Moreover, it has been demonstrated that adipose expression of adiponutrin is decreased by insulin deficiency and in fat-specific insulin receptor knockout mice and increased by insulin replacement in streptozotocin-induced diabetic mice (6). However, our data show a positive correlation between adiponutrin expression and serum insulin levels, in Zucker rats, only when considering the IWAT depot. The results obtained here for leptin were as expected (10) because no change was observed in the obese Zucker rats in response to changes in feeding conditions.

To assess whether the alteration in adiponutrin expression in obese Zucker rats was due to the mutation in leptin receptor or to other metabolic alterations associated with the obese state such as chronic hyperinsulinemia, we studied Wistar rats fed with a high-fat diet for 6 months. It has been described that a long-term consumption of a fat-rich diet induces overweight and insulin resistance in Wistar rats (25). In fact, our rats attained 11% overweight and were hyperinsulinemic in comparison with lean Wistar rats of the same age.

Surprisingly, our results show that adiponutrin expression levels in Wistar rats fed a hyperlipidic diet are much lower in the different adipose tissue depots (except in the IWAT one) than those found in lean Wistar rats. These results could be due to an adaptation to a long-term high-fat diet that would be in agreement with the reported regulation of adiponutrin expression that is dependent on the type of macronutrient (3, 4, 7). In fact, fatty acids, contrary to carbohydrates or proteins, have no stimulatory effect on adiponutrin expression after fasting, and similar results have been obtained in vitro (4, 7). Interestingly, when we compare adiponutrin expression in the different depots in these overweight rats, it is observed that the expression is increased in the IWAT depot, reaching the same values as in the other white adipose tissue depots; this is coincident with what happens in obese Zucker rats. Therefore, it seems that in an overweight or obese condition adiponutrin is overexpressed in the subcutaneous adipose tissue, which is the same as what happens for leptin expression.

When studying the effect of feeding conditions, similar to what happened in obese Zucker rats, in overweight Wistar animals, the decrease in adiponutrin expression induced by fasting was also slight in comparison with what happened in lean animals; but, unlike obese Zucker, the initial levels were recovered after refeeding. It is of note that here, contrary to previous reports (3, 4), we find a stimulatory effect of refeeding with a high-fat diet, and this is probably because the results are not the reflection of an isolated administration of fat after fasting, but the animals had been adapted to this diet for a long time. However, an effect of the different age of the animals (overweight and lean Wistar rats were 6.7 and 3 months old, respectively) in adiponutrin expression levels or in its response to feeding conditions cannot be discarded. Regarding leptin, contrary to adiponutrin, their mRNA levels were not as dramatically affected by feeding conditions in overweight rats, thus indicating some differences in the regulation of both proteins.

These results obtained for adiponutrin in overweight Wistar and in obese Zucker rats, taken together, indicate that their response to fasting is impaired. The lack of response to refeeding in the obese Zucker rats could be due to the higher hyperinsulinemia in these animals but could also indicate a possible role of leptin receptor in adiponutrin regulation. The implication of leptin signaling in adiponutrin regulation, although needing further investigation, could explain the similarities in the regulation of the expression of both proteins. Our results agree with a recent study reporting that obese subjects that are insulin resistant fail to up-regulate the adiponutrin gene (7).

Although the exact physiological role of adiponutrin still remains unknown, our results support that acute energy changes modulate the adipose expression of adiponutrin deeper and faster in comparison with the expression of other adipokines, such as leptin, so adiponutrin could be considered an acute nutritional sensor (3, 26, 27). Moreover, as we describe here that adiponutrin regulation by feeding is altered in the obese state, this protein could be an interesting tool to study, in these models, alterations in the response to signals regulating energy metabolism and homeostasis and, all things considered, to better understand the problem of obesity. Therefore, here we propose adiponutrin as a marker to go further into the research of the cause(s) of the metabolic alterations in response to feeding conditions produced in obesity.

In summary, we report that adiponutrin expression is differently expressed in the different adipose tissue depots, its expression is under tight nutritional regulation in all of them, and its regulation by feeding is altered in the obese state. Because animals lacking functional leptin receptor respond poorly to refeeding, insulin, and also leptin signaling, could be necessary for the adiponutrin regulatory pathway.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by the Spanish Government (Grants G03/028, BFI2003-04439 and AGL 2004-07496/ALI). Our laboratory is a member of the European Research Network of Excellence NuGO (The European Nutrigenomics Organization, European Union Contract FOOD-CT-2004-506360 NUGO). A.C. is a recipient of a fellowship from the Government of the Balearic Islands.

Footnotes
  • 1

    Nonstandard abbreviations: EWAT, epididymal white adipose tissue; IWAT, inguinal white adipose tissue; MWAT, mesenteric white adipose tissue; RWAT, retroperitoneal white adipose tissue; IBAT, interscapular brown adipose tissue; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); ELISA, enzyme-linked immunosorbent assay.

  • The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
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