There is increasing evidence that poor early growth confers an increased risk of type 2 diabetes, hypertension, and other features of the metabolic syndrome in later life. We hypothesized that this may result from poor nutrition during early life exerting permanent effects on the structure and function of key metabolic organ systems. To study the long-term impact of early-life undernutrition on susceptibility to visceral adiposity, we used a rat model of maternal protein restriction (MPR) in which dams were fed a low-protein diet (containing 8% instead of 20% protein in control diet) throughout pregnancy and lactation. MPR offspring were born smaller than controls (offspring of dams on control diet) and in adulthood developed visceral adiposity. We compared the pattern of gene expression in visceral adipose tissue (VAT) between MPR offspring and controls with Affymetrix rat expression arrays. Of the total number of genes and expressed sequence tags analyzed (15,923 probe sets), 9,790 (61.5%) were expressed in VAT. We identified 650 transcripts as differentially expressed ≥1.5-fold in the VAT of MPR offspring. Gene ontology analysis revealed a global upregulation of genes involved in carbohydrate, lipid, and protein metabolism. A number of genes involved in adipocyte differentiation, angiogenesis, and extracellular matrix remodeling were also upregulated. However, in marked contrast to other rodent models of obesity, the expression of a large number of genes associated with inflammation was reduced in this rat model. Thus visceral adiposity in this early-life programmed rat model is marked by dynamic changes in the transcriptional profile of VAT. Our data provide new insights into the molecular mechanisms that underlie the early-life programming of visceral adiposity.
- visceral adipose tissue
- maternal protein restriction
- DNA microarray
obesity is a serious medical problem not only because it substantially impairs quality of life but also because it increases the risk of hypertension, type 2 diabetes, coronary heart disease, sleeping disorders, and cancers (43). There is strong evidence for a genetic component to human obesity (28). Multiple systems regulate energy homeostasis (35, 44), and a number of genes associated with human obesity have been identified (19), yet the genetic component of this condition cannot explain the dramatic increase in the prevalence of obesity in recent years.
A large number of epidemiological studies have revealed a strong statistical association between poor fetal growth and the subsequent development of type 2 diabetes, hypertension, and obesity, visceral obesity in particular (54). These observations were made initially by Barker et al. in England (3a) but have now been reproduced in a large number of populations worldwide. These findings have led to the “fetal origins” hypothesis, which states that an adverse intrauterine environment programs or imprints the development of fetal tissues, permanently determining physiological responses and ultimately producing dysfunction and disease (60). However, the molecular mechanisms that underpin this relationship remain elusive.
In an attempt to provide a conceptual framework to begin to explain these observations, the thrifty-phenotype hypothesis has been proposed (24), which postulates that fetal development is sensitive to the nutritional environment. When it is poor, an adaptive response is initiated to optimize the growth of certain organs, such as the brain, at the expense of others, such as peripheral organs. These adaptations serve to improve chances of fetal survival. They also lead to an altered postnatal metabolism, which enhances postnatal survival under conditions of intermittent and poor nutrition. However, these adaptations would become detrimental if postnatal nutrition were adequate or overabundant. The detrimental consequences may include increased risk of developing obesity and the associated metabolic diseases. Indeed, epidemiological studies in the human have suggested that adults who were growth restricted in utero (occurs in 5–10% pregnancies) have increased body fat and in particular increased visceral fat (52, 61).
A wide variety of animal models have been established to test the fetal origins hypothesis (55). Among them, the maternal protein restriction (MPR) rat model has been used extensively to study the long-term impact of an adverse intrauterine environment on susceptibility to hypertension, insulin resistance, type 2 diabetes, and other metabolic diseases (55). In this model, rat dams are subjected to a low-protein diet containing 8% instead of 20% protein (control diet) throughout pregnancy and lactation. The MPR offspring are known to exhibit low birth weight, the characteristic feature of intrauterine growth restriction, and become diabetic, insulin resistant, and hypertensive in adulthood (56). Other models of fetal programming also predispose to the development of these metabolic disorders, including maternal caloric restriction, uterine artery ligation, and excessive fetal glucocorticoid exposure (55). It is remarkable that these different insults in fetal life produce the same detrimental consequences that occur in adulthood, suggesting that a common mechanism may underlie the early-life programming of the adult diseases.
However, few studies have focused on the long-term impact of an adverse intrauterine environment on the susceptibility to obesity (57) and none on the early-life programming of adipose tissue gene expression. Therefore, the aims of the present study were to determine, using the MPR rat model, 1) whether poor early nutrition led to the development of visceral adiposity and 2) whether the phenotypic changes in fat mass were associated with alterations in the transcriptional profile of visceral adipose tissue (VAT).
MATERIALS AND METHODS
Animals and dietary manipulations.
Virgin female Wistar rats (initial weight 240–260 g) were purchased from Charles River Laboratories (Wilmington, MA) and bred in our animal care facility (Lawson Health Research Institute). They were housed individually and maintained at 22°C on a 12:12-h light-dark cycle. They were mated, and day 0 of gestation was set as the day on which vaginal plugs were expelled. Pregnant rats were fed a diet containing 20% protein (control) or an isocaloric diet containing 8% protein (low protein) throughout pregnancy and lactation. The composition and source of the diets are described in detail in Table 1. There were no differences in litter size or sex ratio between the control and protein-restricted groups. Litters from four control and four protein-restricted dams were followed in this study. At 3 days of age, litters were randomly reduced to eight pups, thus ensuring a standard litter size per dam. At 21 days of age, all pups were weaned onto a 20% protein diet. For simplicity, the two groups of offspring will be termed control and MPR rats. For consistency, only male offspring were used for the study because early-life programming is known to occur in a sexually dimorphic manner (41), which was not the focus of this study. At 130 days of age, male offspring were killed and their visceral fat pads (composed of mesenteric, omental, and retroperitoneal fat masses) isolated, weighed, frozen on dry ice, and stored at −80°C until use. This age was chosen because these animals were monitored for a separate study, after weaning, for their status of insulin homeostasis and found to exhibit insulin resistance for the first time (data not shown).
Paraffin-embedded sections (5 μm) of fat pad from control and MPR rats were stained with Oil red O. Sections were examined under a standard microscope, and the photomicrographs were captured at ×20 magnification.
Total RNA was extracted from homogenized adipose tissues with TRIzol (Invitrogen Life Technologies, Burlington, ON, Canada) and subsequently purified by RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada) coupled with on-column DNase digestion with the RNase-Free DNase Set (Qiagen) according to the manufacturer's instructions.
DNA microarray and data analysis.
A total of four arrays (Affymetrix Rat Expression Array RAE230A; Affymetrix, Santa Clara, CA) were conducted using total RNA samples from two control and two MPR rats. Microarrays were performed at the London Regional Genomics Centre (London, ON, Canada) following the standard procedures as outlined in the Affymetrix GeneChip Expression Analysis Technical Manual. The complete data set was submitted to the National Center for Biotechnology Information's Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) database (acc. no. GSE1813).
Expression values for the Affymetrix GeneChip data were globally normalized to a preset value and analyzed using GeneSpring version 5.0 software (Silicon Genetics, Redwood City, CA). Values for the mean expression level for each gene were calculated for the control and MPR microarray data sets. Candidate genes were selected by a combination of absolute analysis (the absolute call was present on one of the two control arrays and on one of the two MPR arrays for genes considered to exhibit decreased and increased expression, respectively) and comparison analysis (displayed >1.5-fold increase or decrease from the control to MPR animals). A total of 15,923 data sets were sorted according to the following stringent criteria. To reduce false positives, data sets were excluded if 1) the raw values displayed >10-fold difference between the two data sets in either group (17 datasets were excluded); 2) one of the raw values in the two MPR datasets was less than the mean raw value of the two control data sets for genes exhibiting increased expression (15 data sets were excluded); and 3) one of the raw values in the two control data sets was less than the mean raw value of the MPR data sets for genes exhibiting decreased expression (14 data sets were excluded). This resulted in 650 genes being identified as differentially expressed in MPR offspring compared with the controls.
Real-time quantitative RT-PCR.
Real-time quantitative RT-PCR (qRT-PCR) was used to study the expression of seven representative genes identified as differentially expressed (displayed variable degrees of change ranging from −4.7- to +3.3-fold) by the DNA microarray. In addition, transcript levels of two genes that displayed no change in expression with DNA microarray were also determined. Another important consideration for choosing these genes for qRT-PCR was their established role in adipogenesis, angiogenesis, or obesity. qRT-PCR was performed on the same total RNA samples as those used in the DNA microarray study and on additional RNA samples from four control and four MPR rats. Thus a total of six RNA samples per group were subjected to a two-step customer-designed SYBR Green I chemistry-based qRT-PCR, as described previously (71).
Briefly, 1 μg of total RNA was reverse transcribed in a total volume of 20 μl with the High Capacity Complementary Deoxyribonucleic Acid (cDNA) Archive Kit (Applied Biosystems), following the manufacturer's instructions. For every RT reaction set, one RNA sample was set up without reverse transcriptase enzyme to provide a negative control. Gene-specific primers (detailed in Table 2) were designed using Primer Express Software (Applied Biosystems, Foster City, CA), and the optimal concentrations for each gene were determined empirically. All primers were purchased from Sigma Genosys (Oakville, ON, Canada). The SYBR Green I assay was performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a modified universal thermal cycling condition (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 10 s each at 95, 60, and 72°C) with the standard disassociation/melting parameters (15 s each at 95, 60, and 95°C) on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The specificity of the SYBR Green I assay was verified by performing a melting curve analysis and by subsequent sequencing of the PCR products.
Levels of 28S rRNA (housekeeping gene) and target mRNAs in each RNA sample were quantified by the relative standard curve method (Applied Biosystems). Briefly, standard curves for 28S rRNA and each target gene were generated by performing a dilution series of a mixed cDNA pool. For each RNA sample, the amount of target mRNA relative to that of 28S rRNA was obtained. For each target gene, fold changes in the MPR group compared with the control were then calculated and expressed as means ± SE. Data were analyzed by a standard t-test, and significance was set at P < 0.05. All calculations were performed using SPSS software version 9.0 (Chicago, IL).
MPR leads to growth restriction and visceral adiposity.
The male offspring of dams fed a low-protein (8%) diet were significantly smaller at birth than offspring of control dams fed a diet containing 20% protein (5.01 ± 0.15 vs. 6.40 ± 0.15 g, P < 0.01). At 130 days of age, the mean body weight of MPR male offspring was slightly lower than that of the controls, but the weight of visceral fat mass as well as the ratio of visceral fat to body weight were significantly higher (Fig. 1), indicative of visceral adiposity. As shown in Fig. 2, the sizes of adipocytes were similar between control and MPR rats, suggesting that visceral adiposity in this rat model was primarily a result of hyperplasia.
Overview of changes in VAT gene expression.
To determine whether MPR alters the transcriptional profile of VAT and to gain insight into the identity of genes and molecular pathways involved in the pathogenesis of visceral adiposity, DNA microarray experiments were conducted on VAT derived from MPR and control rats. Of the 15,923 genes and expressed sequence tags (ESTs) analyzed (hereafter referred to as genes), 9,790 (61.5%) were expressed at sufficient levels for detection in VAT. MPR altered the expression of 650 genes (>1.5-fold induction or repression) in VAT. Of these, 376 are known genes, including ESTs displaying sequence homology to known genes (Table 3). Among these 376 genes, 57 are of unknown function. However, 87 genes were associated with metabolism, including carbohydrate, lipid, protein, and other metabolic processes. Importantly, 78% (68 of 87 genes) were upregulated by MPR (Table 3). Moreover, 50 genes were classified as involved in inflammation, all but two of which (96%) were downregulated by MPR. In addition, 9 of 10 genes involved in cell cycle regulation were also suppressed. A total of 120 genes were differentially expressed twofold or more in the MPR group (Tables 4 and 5).
MPR increases expression of genes associated with carbohydrate metabolism.
The expression of several genes involved in carbohydrate metabolism was upregulated by MPR (Table 3). Expression of the gene encoding glucose transporter-4 (GLUT4, +1.6-fold), which plays a key role in glucose uptake by adipocytes (12), was upregulated. MPR also increased the expression of glucose-6-phosphate isomerase (+1.6-fold) and phosphofructokinase (+1.5-fold), two important regulators of the glycolytic pathway (65). The expression of both malic enzyme (+1.8-fold) and malate dehydrogenase (+1.6-fold), two critical enzymes responsible for linking glycolysis with the tricarboxylic acid (TCA) cycle (65), was upregulated by MPR.
MPR increases expression of genes associated with lipid metabolism.
Several genes involved in lipogenesis or adipocyte differentiation were upregulated by MPR (Table 4). Expression of fatty acid synthase (FAS, +3.3-fold), a key lipogenic enzyme (40), was upregulated by MPR. Moreover, MPR also increased the expression of other lipogenic enzymes, including glycerol-3-phosphate dehydrogenase [G3PDH, +1.9-fold (65)], acetyl-CoA carboxylase [ACC, +1.7-fold (65)], and the enzyme that catalyzes the rate-limiting step in lipogenesis, stearoyl-CoA desaturase [SCD, +2.6-fold (50)]. In addition to linking glycolysis with the TCA cycle, malic enzyme (+1.8-fold) also plays an important role in generating NADPH necessary for lipogenesis (65). It is well known that ANG (ANG) II is involved in lipogenesis (2), and the intracellular level of ANG II can be augmented either by the increased conversion of ANG I to ANG II or by the decreased inactivation of ANG II. It was remarkable that MPR not only upregulated the expression of chymase-1 (+2.0-fold), an enzyme involved in the conversion of ANG I to ANG II (15), but also downregulated the expression of leucyl-specific aminopeptidase (−1.8-fold), an enzyme that inactivates ANG II (73). Thus the expression of genes altered by MPR suggested the promotion of lipogenesis.
However, the expression of sterol regulatory element-binding protein-1c/adipocyte determination differentiation-dependent factor 1 (SREBP-1c/ADD1), a critical transcription factor involved in positively regulating a number of lipogenic enzymes (30), was not altered by MPR, suggesting that factors other than SREBP-1c may be responsible for the increased expression of lipogenic enzymes in the MPR adipose tissue. One such factor may be Spot-14, a gene known to activate the expression of lipogenic enzymes (14) and the expression of which (+1.6-fold) was upregulated by MPR.
In addition to upregulating genes associated with lipogenesis, MPR also enhanced the expression of genes involved in the uptake of free fatty acids (FFA) from circulation, including very low-density lipoprotein (VLDL) receptor [+1.6-fold (21)] and LDL-related protein-1 [+1.6-fold (6)]. Lipoprotein lipase, an enzyme responsible for hydrolyzing circulating lipids to produce FFA available for cellular uptake (74), and hormone-sensitive lipase, which plays a vital role in the mobilization of FFA from adipose tissue by controlling the rate of lipolysis of the stored triglycerides (23), were expressed abundantly in VAT, but MPR did not influence their expression. Digestion of dietary fat occurs through the activation of two enzymes: gastric lipases, secreted in the stomach, and pancreatic lipases, secreted in the duodenum. Interestingly, expression of pancreatic lipase [+4.6-fold (16)] and its activating cofactor, colipase [+7.0-fold (72)], was markedly upregulated by MPR. The expression of these two genes is novel to VAT, where they may play a role in facilitating the uptake of lipids from circulation and/or in cellular lipolysis. MPR also increased the expression of enzymes involved in cholesterol synthesis, sterol C5-desaturase [+1.6-fold (49)] and isopentenyl-diphosphate Δ-isomerase [+1.5-fold (5)]. Thus both cholesterol uptake and synthesis appeared to be enhanced by MPR.
MPR alters expression of genes associated with protein metabolism and other metabolic processes.
Numerous pancreatic protein metabolizing enzymes, whose expression is novel to adipose tissue, including pancreatic cationic trypsinogen, pancreatic trypsin-1 (70), chymotrypsin-like chymotrypsin B (4), and carboxypeptidase B (13), were upregulated by MPR (Table 4). These pancreatic secreted enzymes normally function to digest proteins in the small intestine. In adipose tissue they may be involved in extracellular matrix (ECM) remodeling, as demonstrated in various carcinomas (59). MPR also altered the expression of enzymes involved in a variety of other metabolic processes, including nucleic acid metabolism, steroid hormone metabolism, and drug detoxification (Table 3).
MPR increases expression of genes associated with cellular proliferation and differentiation.
MPR increased the expression of a number of growth factors, which are known to influence cellular proliferation and differentiation. These included transforming growth factor-α [TGF-α, +2.3-fold (66)], bone morphogenetic protein-3 [BMP3, +1.9-fold (3)], connective tissue growth factor [CTGF, +1.8-fold (48)], insulin-like growth factor II [IGF-II, +1.5-fold (39)], and fibroblast growth factors 7 and 21 [+1.8- and +1.5-fold, respectively (53)]. Moreover, several factors involved in adipocyte differentiation were upregulated by MPR, including MAP kinase phosphatase-1 [MKP-1, +2.2-fold (64)] as well as transcription factors CCAAT box enhancer-binding protein (C/EBP)-δ and C/EBP-β [+1.9- and +1.6-fold, respectively (22)].
MPR alters expression of genes associated with ECM remodeling.
MPR increased the expression of matrix metalloproteinase (MMP)-24 (+1.7-fold), which functions to activate MMP-2 (62), an enzyme known to play an important role in adipose tissue ECM remodeling. The expression of the ECM proteoglycans tetranectin [+8.7-fold (76)] and fibromodulin [+1.8-fold (32)], which are involved in cell-cell adhesion and participate in the assembly of the ECM, was upregulated by MPR. There was also a marked downregulation of genes encoding ECM components, including osteopontin (−5.2-fold), fibrillin (−4.0-fold), glycoprotein nmb (−3.6-fold), cadherin-22 (−2.7-fold), and type-1 collagen (−1.8-fold) (59). In addition, expression of cystatin N [−8.6-fold (1)], which functions as an endogenous inhibitor of the lysosomal cysteine proteinases, was profoundly downregulated.
Interestingly, expression of genes encoding a range of proteins associated with cytoskeletal structure of cells was upregulated by MPR. These included actin (+7.6-fold), myosin light chain-1 (+2.1-fold), myosin light chain-2 (+1.7-fold), tropomyosin (+1.8-fold), integrin (+1.9-fold), and the integrin-complexing protein Tspan-2 (+1.5-fold). Moreover, MPR increased the expression of several neuron-related structural proteins, including myelin protein zero (+4.5-fold), proteolipid protein (+2.1-fold), and limbic system-associated membrane protein (+1.9-fold). Therefore, these complex alterations in the expression of many ECM proteins and enzymes were indicative of increased ECM remodeling activities in the adipose tissue of MPR animals.
MPR enhances expression of angiogenic factors.
Accumulating evidence suggests that adipose tissue growth/expansion is dependent on angiogenesis. The expression of several proangiogenic factors was upregulated by MPR. These factors included leptin [+1.9-fold (7)], CTGF [+1.8-fold (8)], cysteine-rich protein-61 [CYR61, +1.8-fold (8)], and dermatopontin [+1.6-fold (51)]. In addition, MPR increased the expression of chymase-1 (+2.0-fold), a proangiogenic enzyme involved in the conversion of ANG I to ANG II (15). The angiogenic effects of ANG II in adipose tissue were likely further enhanced in MPR animals, since the expression of leucyl-specific aminopeptidase (−1.8-fold), an extracellular enzyme that inactivates ANG II (73), was decreased. In contrast, MPR caused downregulation of the two antiangiogenic factors, F-spondin [−2.6-fold (69)] and properdin [−2.6-fold (42]). Thus MPR altered the pattern of adipose tissue gene expression in favor of angiogenesis.
MPR decreases expression of genes associated with cell-cycle regulation.
Bcl-2-related protein-A1 is involved in regulating cell-cycle progression, and its expression (−2.8-fold) was repressed by MPR. In addition, several other genes involved in regulating the cell cycle include mitogen-inducible gene 2 (−2.7-fold), cyclin-dependent kinase (−2.3-fold) and cell division cycle 25B (−1.5-fold) were also downregulated by MPR.
MPR suppresses expression of genes involved in inflammation.
The most notable class of genes downregulated by MPR was that associated with inflammation (Tables 3 and 5). The expression of several immunoglobulin and antigen genes was decreased (Table 5). Glycosylation-dependent cell adhesion molecule 1 (Glycam-1) is an endothelial cell glycoprotein that functions as an adhesive ligand for the lymphocyte adhesion molecule, l-selectin. Glycam-1 (−6.5-fold) expression was downregulated dramatically by MPR. Expression of 12-lipoxygenase, one of the key enzymes involved in arachidonic acid metabolism, was decreased more than fourfold (Table 5). Expression of two inflammatory proteins, S100 calcium-binding protein-A8 (−2.7-fold) and S100 calcium-binding protein-A9 (−2.2-fold), which form functional heterodimers, was reduced by MPR. The expression of sialophorin (−3.3-fold), a member of a ligand receptor complex involved in T cell activation, was also downregulated. Moreover, MPR downregulated the expression of complement component 3A (−1.8-fold) and 8 (−1.5-fold), which are members of the complement immunity pathway.
Only three inflammation-related genes were upregulated by MPR, and they were NF-κB inhibitor-α (IκBα, +2.1-fold), FK506-binding protein-2 (+1.9-fold), and CD1D (+1.8-fold). Two of the three upregulated genes, IκBα and FK506-binding protein-2, are negative regulators of inflammatory processes. Thus MPR altered the expression of genes associated with inflammation in a manner compatible with a suppressed inflammatory state.
Confirmation of microarray data by qRT-PCR.
To validate the DNA microarray data, we determined the expression of several candidate genes that exhibited increased or decreased expression by microarray by use of qRT-PCR. In addition, the expression of two genes that displayed no change in expression by microarray was also examined. qRT-PCR confirmed that the data from the gene chips were robust and that in many cases the magnitude of fold changes was underestimated on the microarrays (Fig. 3).
Accumulating evidence suggests that poor early growth is associated with an increased risk of developing insulin resistance, type 2 diabetes, and hypertension in later life (25, 29). Although it is generally accepted that obesity is required for the full expression of this thrifty phenotype (56), very few studies have focused on the long-term impact of poor early growth on the susceptibility to obesity in adult life. Ozanne et al. (57) recently reported the long-term effects of poor early nutrition on weight gain and the development of obesity in response to a highly palatable cafeteria-style diet. They showed that MPR mice at defined time windows not only had long-term (programmed) effects on weight gain but also altered the response to an obesity-inducing cafeteria-style diet and, hence, the susceptibility to obesity in later life. Mice who were growth restricted in utero but underwent rapid postnatal catch-up growth were heavier than control offspring throughout the study. These animals also gained more weight than the control offspring in response to the obesity-inducing diet. In marked contrast, mice that were growth restricted during lactation remained permanently smaller than controls. Moreover, when these mice were weaned onto the obesity-inducing diet, they showed no additional weight gain compared with their littermates on a control diet. These findings suggest that nutritional influences, both pre- and postnatal, can exert long-term (programmed) effects on body weight homoeostasis in later life. However, the molecular mechanisms that underlie this programming effect of poor early nutrition are unknown.
In the present study, we used a similar rat model of MPR in which dams were fed a low-protein diet throughout pregnancy and lactation (33, 58). We chose to study specifically visceral adiposity/obesity because it is the best predictor of the metabolic syndrome (45), which is known to occur in this rat model (55). We demonstrate that MPR predisposes the male offspring to visceral adiposity but not overweight in early adult life. Moreover, we show that MPR dramatically alters the transcriptional profile of VAT.
The cellular development associated with adipose tissue expansion involves adipocyte hypertrophy (increase in cell size) and/or adipocyte hyperplasia (increase in cell number) (26). Hypertrophy is the result of excess triglyceride accumulation in preexisting adipocytes due to a positive energy balance (energy intake in excess of energy expenditure). Hyperplasia, also known as adipogenesis, results from the recruitment of new preadipocytes from progenitor cells in adipose tissue. In addition, it involves the proliferation of preadipocytes and their differentiation into lipid filled mature adipocytes.
Two processes contribute to lipid accumulation in adipocytes, increased lipogenesis and reduced lipolysis. The increased expression of genes encoding a number of lipogenic enzymes (e.g., FAS, G3PDH, ACC, and SCD) in the MPR adipose tissue suggested that enhanced lipogenesis was one of the factors contributing to visceral adiposity in this rat model. Furthermore, an enhanced ability to take up FAA from circulation resulting from increased expression of VLDL receptor and its functionally related protein, as well as an increased capacity for cholesterol synthesis as a result of increased expression of genes coding for enzymes involved in the biosynthesis of cholesterol, also likely contributed to the excess adipose tissue expansion in MPR rats.
In addition, increases in the expression of genes encoding several growth factors known to be involved in regulating cell proliferation and differentiation suggested that an enhanced adipogenesis program was likely a contributing factor in the MPR-induced programming of visceral adiposity. The observed upregulation of MKP-1, a recently identified factor essential for in vitro adipocyte differentiation (64), as well as increased expression of the key transcription factors C/EBP-δ and C/EBP-β involved in promoting adipocyte differentiation, are consistent with this notion. It was shown previously that a large number of cell cycle-related genes were repressed during in vitro adipogenesis (68). It is interesting to note that an equally extensive list of genes associated with cell cycle regulation was downregulated in adipose tissue of MPR rats, adding further support to the notion that adipogenesis was enhanced in this rat model. Moreover, there were no apparent differences in the size of adipocytes between control and MPR rats. Collectively, these findings suggested that the MPR-induced visceral adiposity was primarily a result of adipocyte hyperplasia.
Adipocyte differentiation in vitro involves remodeling of the ECM (22). ECM remodeling is a complex and dynamic process that involves alterations in the expression and activity of ECM remodeling enzymes and the subsequent modification to ECM components. MMP-2 is known to be upregulated in obesity and plays a critical role in adipose tissue ECM remodeling (11, 38). Expression of MMP-24, an enzyme involved in the activation of MMP-2 (62), was increased in MPR rats. Furthermore, MPR upregulated the expression of tetranectin, a plasminogen-binding protein that has been suggested to play an important role in tissue remodeling due to its ability to stimulate plasminogen activation and its expression in developing tissues such as the bone and muscle (31). In addition, expression of genes encoding a large number of cellular structural proteins and pancreatic proteases was also upregulated in MPR rats. Therefore, active adipose tissue remodeling was likely a major factor contributing to the development of visceral adiposity in this rat model.
In addition to promoting adipocyte differentiation, ECM remodeling is also involved in angiogenesis (10). Recent evidence suggests that adipose tissue expansion is angiogenesis dependent (63). It is believed that the angiogenic switch is tightly controlled by a balance between pro- and antiangiogenic factors (17). Thus angiogenesis occurs in a tissue when levels of proangiogenic factors are increased while those of antiangiogenic factors are reduced (18). In the present study, we observed coordinated upregulation of angiogenic factors (leptin, TGF-α, CTGF, CYR61, dermatopontin, and chymase-1) and downregulation of antiangiogenic factors (leucyl-specific aminopeptidase, F-spondin and properdin) in MPR rats. This suggested that angiogenesis likely played a key role in the pathogenesis of visceral adiposity in this rat model. It is noteworthy that the majority of these factors whose expression was altered by MPR have not been described previously in adipose tissue, thus attesting to the utility of this functional genomics approach in identifying new players involved in adiposity/obesity.
In several rodent models of obesity, adipose tissue gene expression profiling has revealed a global upregulation of genes associated with inflammation (46, 47, 67). Adipocytes are capable of expressing inflammatory genes (34). In addition, inflammatory cells such as macrophages have been demonstrated to be present in higher numbers in adipose tissue in obesity (75, 77). It was surprising that a large number of genes involved in inflammatory processes were downregulated in this rat model. Although the precise reasons for this disparity are not apparent at the present time, it is tempting to speculate that the suppressed inflammatory state in the MPR adipose tissue may represent an earlier event occurring during the development of adiposity/obesity, because it is believed that the increased inflammatory response is likely a result of obesity (77). Consequently, it has been suggested that this enhanced inflammatory response may link obesity to its associated metabolic diseases (36). However, the pathophysiological significance of a suppressed inflammation state within the adipose tissue remains to be determined.
Interestingly, several of the changes in the transcriptional profile of VAT as a result of MPR are similar to those observed in white adipose tissue following long-term caloric restriction (27). These include enhanced expression of genes involved in regulating lipogenesis and adipocyte differentiation as well as a global downregulation of genes associated with inflammatory processes. This similarity between the two distinct models suggests that MPR and caloric restriction alter adipose tissue gene activity in a similar way, thus underscoring the significance of our present findings utilizing this rat model in understanding the long-term effects of poor early nutrition on susceptibility to visceral adiposity/obesity in later life.
It is noteworthy that the present study, like many others in the literature (20, 37, 46, 47, 67), used RNA samples derived from adipose tissue for gene expression profile analysis. This kind of study design is distinct (in the objectives and, hence, the interpretations of the results) from those in which RNA extracts prepared from a pure population of adipocytes was utilized (68). Consequently, many of the alterations in adipose tissue gene expression that we observed in this study could be explained by a change in the ratio of adipocytes to other cell types between control and MPR rats. Obviously, the validity of this contention requires future scrutiny.
In conclusion, we demonstrate that maternal protein restriction during pregnancy and lactation programs the susceptibility to visceral adiposity in the adult rat offspring, and that the coordinated upregulation of molecular pathways involved in adipogenesis and angiogenesis likely plays a key role in this process (Fig. 4).
This work was supported by grants from the Canadian Institutes of Health Research.
We thank Brenda Strutt for performing fat tissue histology.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 by American Physiological Society