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Biochemical, Molecular, and Genetic Mechanisms

Energy Restriction Lowers the Expression of Genes Linked to Inflammation, the Cytoskeleton, the Extracellular Matrix, and Angiogenesis in Mouse Adipose Tissue¹

Yoshikazu Higami^*,,**,2, Jamie L. Barger^,**,2, Grier P. Page, David B. Allison, Steven R. Smith, Tomas A. Prolla and Richard Weindruch^,**,#,3

^* Department of Pathology and Gerontology, Nagasaki University Graduate School of Biomedical Science, Nagasaki 852-8523, Japan; Wisconsin National Primate Research Center, Madison, WI 53715; ^** Department of Medicine, University of Wisconsin-Madison, WI 53705; Department of Biostatistics, Section on Statistical Genetics, University of Alabama, Birmingham, AL 35249; Pennington Biomedical Research Center, Baton Rouge, LA 70808; Departments of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, WI 53706; and ^# Geriatric Research, Education and Clinical Center, W.S. Middleton Memorial Veterans Administration Medical Center, Madison, WI 53705

³ To whom correspondence should be addressed. Email: rhweindr{at}wisc.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Using high-density oligonucleotide microarrays, we examined the actions of energy restriction (ER) on the expression of >11,000 genes in epididymal white adipose tissue (WAT) of 10- to 11-mo-old male C57Bl6 mice. Four groups were studied: controls not subjected to food restriction (CO), food-restricted 18 h before being killed (FR), short-term ER for 23 d (SER), and long-term ER for 9 mo (LER). As we reported previously, compared with CO mice, FR and SER minimally influenced the gene expression profiles; however, 345 transcripts of 6266 genes determined to be expressed in WAT were significantly altered by LER. We focus here on the 109 (31%) of these genes that were involved in either inflammation (56 genes), cytoskeleton (16 genes), extracellular matrix (23 genes), or angiogenesis (14 genes). Among these 109 genes, 104 transcripts (95%) were downregulated by LER. Western blotting for heat shock protein 47 and osteonectin, and immunohistochemical staining for hypoxia inducible factor (HIF)-1), supported the microarray data that LER downregulated the expressions of these genes. Additionally, a 75% reduction in adipocyte size with LER reflected the change in the expression of genes involved in cell morphology. Our findings provide evidence that LER suppresses the expression of genes encoding inflammatory molecules in WAT while promoting structural remodeling of the cytoskeleton, extracellular matrix, and vasculature. These alterations may play an important role in the protection against WAT-derived inflammation and in lifespan extension by LER.

KEY WORDS: • microarray • energy restriction • dietary restriction • aging

Aging is associated with obesity, alterations in body fat distribution, insulin resistance and leptin resistance (1–4). Surgical removal of visceral fat prevents obesity-induced and age-associated insulin resistance in rats (5,6). Moreover, recent evidence suggests that white adipose tissue (WAT)⁴ may contribute to the regulation of longevity because mice engineered to have less WAT live longer than their controls (7,8). Several bioactive WAT-derived secretory molecules such as leptin (9), tumor necrosis factor- (10), interleukin (IL)-6 (10), adiponectin (11), and resistin (12) were characterized recently, and some of these molecules are involved in obesity and insulin resistance. Obesity-related cardiovascular disease can be explained in part by disturbances in the hemostatic and fibrinolytic systems because obesity is associated with higher levels of WAT-derived IL-6, C-reactive protein, fibrinogen, factor VII, factor VIII, von Willebrand factor, and plasminogen activator inhibitor-1 (13). Therefore, accumulation and dysfunction of WAT and subsequent derangement of WAT-derived secretory molecules may be major causes of type 2 diabetes, obesity-related cardiovascular disease, and short lifespan (7,8,13).

Long-term energy restriction (LER) remains the most robust, reproducible, and simplest experimental manipulation known to extend maximum lifespan and to retard a broad spectrum of age-associated pathophysiology in laboratory rodents (14–16). Attenuation of oxidative and other stresses and reduction of glycemia and insulinemia may be significant factors in the actions of LER, but the exact underlying mechanism is still debatable (16). LER reduces plasma insulin (15) and leptin levels (17) as well as adiposity. Moreover, LER reverses age-associated insulin and leptin resistance, possibly through decreasing adiposity (18,19). Therefore, the beneficial action of LER on the aging process may be mediated in part by the alteration of the amount and functional status of WAT.

To provide a global analysis of gene expression in WAT of ER mice, the gene expression profiles of >11,000 genes in WAT of 10- to 11-mo-old male C57Bl6 mice subjected to 18 h of food restriction (FR), short-term ER for 23 d (SER), and long-term ER for 9 mo (LER) were compared with those of control mice (CO) using high-density oligonucleotide microarrays. As reported previously (20), 6266 of >11,000 genes were determined to be expressed in WAT. According to the statistical filtering, 2, 2, and 345 transcripts were significantly changed by FR, SER, and LER, respectively. These findings suggest that the transcript phenotype of FR and SER differs minimally from that of CO. In contrast, the expression profile of LER is markedly different from that of CO. A previous report focused on 120 of the 345 genes that were associated with metabolism, and 85% (103 of 120) of these genes were upregulated by LER (20). In this report, we present the LER-induced gene expression profiles involved in cytoskeleton, extracellular matrix, angiogenesis, and inflammation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and dietary manipulations. The husbandry and dietary manipulations of mice were described previously (20). Diet composition of both Control and energy-restricted diets is shown in Table 1. Briefly, male C57Bl6 mice (6–7 wk old) were purchased and maintained under specific pathogen–free conditions until they were killed at 10–11 mo by cervical dislocation. The control (CO) and food-restricted (FR) groups were fed 410 kJ semipurified Control diet/wk (90% of the mean ad libitum food intake, Table 1). The CO mice were fed 117 kJ of food for approximately the last 24 h before they were killed. The LER mice were started on ER feeding at 6 wk of age and fed 243 kJ semipurified diet/wk (a 41% ER; Table 1) thereafter. The SER group was started on ER feeding 23 d before they were killed. Leftover food was removed from LER, SER, and FR mice 18 h before they were killed. Therefore, the food intake for the last 24 h before killing was almost equivalent among the LER, SER, and FR groups (67 kJ of food for the last 24 h). Bilateral epididymal WAT depots were isolated, weighed, and frozen in liquid nitrogen for storage at –80°C. All procedures complied with the Institutional Animal Care and Use Committee of the Veteran's Administration Hospital, Madison, WI.

The value of each RNA abundance was automatically calculated in each array with Affymetrix GeneChip analysis suite version 3.3 after scanning. Based on the Affymetrix algorithm, 6266 of >11,000 genes were determined to be expressed in WAT. For the first step in statistical evaluation of the effects of FR, SER, and LER on gene expression levels, we used nonparametric bootstrap hypothesis testing to obtain a frequentist P-value for each gene. Gene expression change was called significant when the P-value was 0.01. In the second step, we calculated a False Discovery Rate (FDR) for multiple comparison adjustments. As reported previously, 6266 genes were filtrated by our statistical criteria (bootstrap P-value 0.01, FDR 10%, and log₂ adjusted ratio of signal intensity 1.5 or –1.5); the data reported in the tables represent mean intensities ± SEM, the percentage change in signal intensity, P-value, and FDR.

    Western blot analysis. Epididymal WAT from additional age- and strain-matched mice was homogenized in buffer (50 mmol/L Tris-HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% SDS, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride) containing 0.05 µL protease inhibitor cocktail (Sigma Chemical) per milligram WAT. The protein content of WAT homogenates was determined using the bicinchoninic acid assay (Pierce) and 75 µg protein/sample was loaded in precast 16% polyacrylamide gels (Invitrogen). After electrophoresis, proteins were transferred to nitrocellulose membranes (Hybond ECL, Amersham) and stained with Ponceau S to confirm equal protein loading. Membranes were then destained with water and blocked with 10% powdered milk for 1 h at room temperature; all washes and incubations were performed using PBS containing 1% Tween-20. Blots were washed, incubated overnight at 4°C with primary antibodies [anti-rat heat shock protein (Hsp)47: Calbiochem-Novabiochem; anti-human osteonectin: Haematologic Technologies], washed, incubated with secondary antibody (1:5000 horseradish peroxidase-linked anti-rabbit IgG) for 1 h at room temperature, and then subjected to a final wash. After application of chemiluminescent detection reagents (Pierce), blots were exposed to film and band intensity was determined using ImageJ software (21). Statistical comparisons of the densitometry data were made with unpaired t tests using Stat View 5.0 (SAS Institute).

    Immunohistochemistry. Samples of epididymal adipose tissue from CO, LER, and genetically obese Lep^ob/ob mice were fixed in buffered formalin, dehydrated, and embedded in paraffin. Tissue sections (5 µm) were incubated with a mouse monoclonal hypoxia inducible factor (HIF-1) antibody (Novus Biologicals) overnight at 4°C, washed, and incubated with biotinylated anti-mouse IgG for 1 h at room temperature. Staining was performed using standard techniques (22), and sections were subsequently immersed in hematoxylin for visualization of unstained nuclei.

    Adipocyte size. Epididymal WAT (50 mg) was sampled from an additional 8 age- and strain-matched mice (n = 4 CO and 4 LER). Tissue was fixed in osmium tetroxide and assayed for adipocyte size as described previously (23). An unpaired t test was used to test for differences in adipocyte size between groups.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Phenotypic changes in WAT with CR. As reported previously, the mass of epididymal adipose tissue was 84% lower in LER than in CO mice (2.04 ± 0.11 vs. 0.32 ± 0.03 g). This decrease was paralleled by a 77% reduction in adipocyte volume in epididymal WAT from LER mice as seen in this study (77 ± 8 vs. 18 ± 2 x 10^–4 µm³; P < 0.0001).

    LER downregulates the expression of many genes involved in inflammation. The expression of 56 genes involved in inflammation was altered by LER. Of these, only 1 gene, adipsin, an adipocyte-derived component of the alternative complement pathway (24), was significantly upregulated (135%). In contrast, the expression of genes encoding several other complement factors, their regulators and receptors, including complement C1qA, B, and C chains, complement component C4, and the complement receptor C1qr1, was downregulated by LER (Table 2).

Acute phase plasma proteins (APP) mobilize a systemic acute phase reaction to protect against a local tissue injury or infection. Most APP production is induced by cytokines (25,26). Three APP (haptoglobin, lipopolysaccharide-binding protein, serum amyloid A3 protein) were expressed in WAT; haptoglobin was the most abundantly expressed, and all were downregulated by LER (Table 2).

Four genes encoding major histocompatibility complex (MHC)-related molecules were downregulated by LER (Table 2). In addition, MHC-II E ß (decreased by 57%, P = 0.016, FDR = 12.7%) and MHC-II H2-IA ß, k haplotype (decreased by 55%, P = 0.012, FDR = 11.2%) approached our statistical criteria and had lower expression in WAT of LER mice.

Lysozyme M and P are involved in host defense and the regulation of inflammation through their protease activity (27). LER markedly suppressed the expression of both lysozyme M (73%) and P (78%). Lysosomal proteases of the cathepsin family play a critical role in antigen processing and in the formation of peptide-receptive dimers during MHC-II–restricted antigen presentation (28,29). LER markedly downregulated the expression of cathepsin S and Z (Table 2). Moreover, alteration in the expression of cathepsin C (decreased by 30%, P = 0.004, FDR = 5.9%) and cathepsin L (decreased by 30%, P = 0.001, FDR = 2.3%) by LER nearly satisfied our statistical criteria.

We observed that LER downregulated the expression of several genes encoding inflammatory cell-specific proteins (Table 2). Several pan-lymphocyte–related, myeloid cell–related, B-cell– or immunoglobulin-related, T-cell–related, macrophage-related, natural killer cell–related and mast cell–related proteins were included and all genes were downregulated by LER. In addition, LER reduced the expression of 6 other inflammation-related genes (Table 2). Among these, annexin A1 was expressed abundantly and LER suppressed the expression markedly. Annexin A1 exerts an antimigratory action in several models of acute and chronic inflammation as an inhibitor of the process of leukocyte extravasation (30).

    LER downregulates the expression of genes involved in cytoskeleton. Genes encoding cytoskeletal proteins including - and ß-tubulin, -actin, and ß-spectrin were abundantly expressed and were all markedly downregulated by LER (Table 3). Additionally, LER altered the expression of 8 genes encoding actin-modulating proteins, and 7 were downregulated by LER.

View larger version (20K): [in this window] [in a new window]   FIGURE 1  Decreased abundance of 2 proteins in WAT of mice CO and LER mice. Values are mean + SEM, n = 4. Asterisks indicate different from CO: *P = 0.01 and **P = 0.03. The blots shown are representative of each treatment group.

    LER downregulates the expression of genes involved in angiogenesis. For genes classified as involved in angiogenesis (Table 5), there was an overall balance between pro- and antiangiogenic changes in gene expression with LER. Six proangiogenic genes were decreased in WAT, including Vegfd (35), Fzd4 (36), Hif1 (35), Gal3 (37), osteonectin (38), and Mest (39). The expression of 5 other genes likely resulted in a net proangiogenic effect, with increased expression of one proangiogenic gene [Idb1, see (35,40)] and decreased expression of 4 genes with antiangiogenic action: Pedf (41), Thbs1 (40), Serpinb6 (42), and Mkbp1 (43). Several angiogenesis-related growth factors almost met the selection criteria and were all downregulated by LER: fibroblast growth factor 1 (decreased by 54%, Fgf1, P = 0.01, FDR = 10.1%), angiopoietin 2 (Ang2, decreased by 41%, P = 0.038, FDR = 20.6%), and platelet-derived growth factor receptor ß (Pdgfrb, decreased by 42%, P = 0.034, FDR = 19%). In addition to downregulating the expression osteonectin, the average level of osteonectin protein was decreased by 95% in WAT of LER mice (Fig. 1). Furthermore, decreased expression of HIF-1 in LER mice was reflected at the protein level as assessed by immunohistochemistry, i.e., nuclei in adipose tissue of LER mice showed minimal HIF-1 staining compared with nuclei in adipose tissue of CO mice, which showed frequent HIF-1 staining (Fig. 2A and B). Nuclei from genetically obese Lep^ob/ob mice, on the other hand, showed intense nuclear staining of HIF-1 (Fig. 2C).

View larger version (77K): [in this window] [in a new window]   FIGURE 2  Immunohistochemical detection of HIF-1 in epididymal WAT of LER (A), CO (B), and genetically obese Lepob/ob mice (C). HIF-1 translocates to the nuclei of cells and the presence of HIF-1 is seen as brown staining (indicated by red arrows); HIF1-negative nuclei (indicated by blue arrows) are blue due to hematoxylin counterstaining. The WAT of LER mice (A) contained almost exclusively HIF-1-negative nuclei, whereas that of CO mice (B) was predominantly HIF-1-positive. Expression of HIF1 was previously shown to be increased in obese mice; nearly all nuclei in WAT of genetically obese Lepob/ob mice showed intense staining for HIF-1 (C). All images are 400X magnification.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In addition to the macrophage-specific genes, many other inflammatory genes downregulated by LER were previously reported to be increased with obesity (46). These include small inducible cytokines A2, A6, and A9, colony stimulating factor 1, cathepsins S and Z, and others. In total, 21 of the 56 genes involved in inflammation that were decreased with LER were the same genes reported to be positively associated with obesity (see Table 2). It is likely that further overlap exists between the genes associated with obesity and the genes downregulated by LER, but we could not identify these genes because the techniques of analyzing the microarray data differed among the studies. Moreover, our previous work using high-density oligonucleotide microarrays in mouse brain (50) and monkey muscle (51) suggested that aging enhanced the expression of genes involved in the inflammatory process, and in brain, LER suppressed the age-related inflammation. Therefore, it is likely that the attenuation of inflammation by LER is a common finding in several organs in addition to WAT.

Adipsin was the only inflammatory gene increased in expression with LER, and this observation is in agreement with decreased adipsin expression in murine obesity (52). In these obese models, however, the defect in adipsin secretion precedes the development of obesity, and it is thought that adipsin levels may not be related to the amount of adipose tissue per se (53). Alternatively, adipsin expression may be regulated by the sympathetic stimulation of WAT as evidenced by increased expression after treatment with a mixture of ephedrine and caffeine (54). We previously reported an increase in the expression of ß-adrenergic receptors in WAT of LER mice (20), and this may explain the increased expression of adipsin with LER. Viewed as a whole, the change in gene expression with LER highlights the growing awareness of the interplay among macrophage biology, inflammation, and metabolism (55).

In addition to being a source of inflammatory cytokines, adipose tissue also secretes other factors that affect whole-body physiology in response to nutritional stimuli (56). Although the mechanism by which LER alters the secretory activity of adipose tissue is not known, one possibility is that LER induces a metabolic shift in adipose tissue that prevents adipocyte hypertrophy and consequently its secretory activity. Using histological methods, we previously found that LER reduced the size of adipocytes (20), and when measured quantitatively, there was a 77% reduction in the size of adipocytes in LER mice. Kadowaki et al. (57) suggested that hypertrophied adipocytes secrete less of the insulin-sensitizing hormone, adiponectin, and have increased secretion of hormones such as resistin, leading to insulin resistance in obesity; in contrast, small adipocytes with less lipid accumulation have increased secretion of adiponectin. In fact, we found that LER increased the expression of adiponectin (20). Similarly, LER significantly increases plasma levels of adiponectin in mice throughout their lives (58). Possibly, the lowered expression of several genes encoding cytoskeletal proteins (e.g., tubulins, actin, spectrin; Table 3) might reflect the changed morphology of adipocytes in LER mice. Similar cytoskeletal alterations occur early in adipocyte differentiation, suggesting that they participate in the alteration of adipocyte morphology and influence subsequent biosynthetic events (59). Therefore, LER might regulate adipocyte bioactivity via cytoskeletal remodeling.

There was a notable overlap in the genes altered with LER and genes involved in adipocyte differentiation. During adipocyte differentiation, an upregulation occurs in the expression of genes encoding metabolism-associated proteins, and we previously observed this to occur with LER (20). In addition, collagen type VI is upregulated but collagen types I and III are downregulated during adipocyte differentiation in vitro (60), and we observed that LER downregulated the expression of procollagens, their modulators and cell adhesion molecules (Table 4). It is generally accepted that extensive extracellular matrix remodeling and modifications of proteolytic activities in WAT occur during the development of obesity. LER-associated extracellular matrix remodeling might also be involved in the modifications of matrix Mmp expression. Mmp alter the integrity of the extracellular matrix and their activity has been implicated in obesity; when fed a high-fat diet, Mmp11-deficient mice developed adipocyte hypertrophy compared with wild-type mice (61), whereas the expression of Mmp12 is strongly induced WAT from obese mice (34). Interestingly, LER enhanced Mmp11 expression and reduced Mmp12 expression (Table 4).

Transforming growth factor ß (Tgfß) inhibits adipocyte differentiation, and that this inhibition is associated with the enhanced expression of procollagen, type 1 1 (62). We observed that LER downregulates Tgfß1-induced transcript 4 (decreased by 42%, P = 0.013, FDR = 11.4%), Tgfß inducible protein and procollagen, type I 1 expressions (Table 4). Therefore, the attenuated expression of collagen by LER might be dependent upon the LER-associated reduction of Tgfß synthesis. Hsp47 encodes a procollagen synthesis-specific molecular chaperone, which is essential for secretion of procollagen from cells (31). Tgfß induces Hsp47 expression during collagen synthesis (63). Lox1 and Lox2 are involved in collagen synthesis as well (32). Therefore, LER results in extracellular matrix remodeling involved in the reduction of collagen synthesis, perhaps via the decreased expression of Tgfß, Hsp47, Lox1, and Lox2.

The WAT is highly vascularized with an extensive capillary network surrounding each adipocyte; it also has angiogenic properties (64). Although the effect of LER on the expression of genes involved in angiogenesis was essentially balanced between pro- and antiangiogenic actions, many other genes with putative proangiogenic activity were decreased with LER, including leptin [decreased 92%, see (20)], Tgfß1 (35) and other cytokines and inflammation-related proteins including Ccl2 (35), haptoglobin (65), and cathepsin S (66). Gross histological examination did not indicate significant alteration of the vasculature in WAT between CO and LER mice, but the level of 1 antiangiogenic protein, osteonectin, was significantly decreased in LER mice. Overall, LER appears to attenuate angiogenesis within WAT.

It is tempting to speculate that the LER-induced changes in WAT size, inflammation, and angiogenesis are functionally interrelated. Such a mechanism was proposed to occur during the development of obesity in which adipocyte hypertrophy leads to localized hypoxia, which in turn elicits an inflammatory response to stimulate angiogenesis within the WAT (44). A key regulator in this feedback loop is the transcription factor HIF-1; its expression is increased in hypoxia and it regulates the expression of many angiogenic genes. This pathway is active and was characterized extensively in tumor growth, but it may also be present in WAT; expression of leptin and Vegf is increased in 3T3-F442A adipocytes cultured in hypoxia, coincident with an elevated accumulation of nuclear HIF-1 protein (67). Conversely, we observed a downregulation of these same genes in LER relative to CO mice in addition to decreased nuclear staining of HIF-1 in LER compared with CO and genetically obese mice. We propose that the prevention of adipocyte hypertrophy with LER attenuates the WAT-derived inflammation by preventing hypoxia within the tissue.

We demonstrate here that only LER, but not FR or SER, induces unique alterations at the transcriptional level in WAT. Therefore, the health benefits of LER may derive from both a reduction in overall adiposity (typically by 70%) and an alteration in its functional characteristics. Notable elements of the latter include an upregulation of genes involved in mitochondrial energy metabolism (20) and suppression of inflammation, altered production of adipocytokines, and the induction of cytoskeletal, extracellular matrix, and vasculature remodeling.

	ACKNOWLEDGMENTS

 
The authors thank Mark Devries for assistance with the immunohistochemical detection of HIF-1.

	FOOTNOTES

 
¹ Supported by National Institutes of Health R01 AG18922 (R.W.), National Institutes of Health T32AG00213 (J.L.B.), and National Science Foundation 0090286 (D.B.A.).

² These authors contributed equally to this work.

⁴ Abbreviations used: APP, acute phase protein; CO, control-fed mice; ER, energy restriction; FR, food-restricted; FDR, false discovery rate; HIF, hypoxia inducible factor; Hsp, heat shock protein; IL, interleukin; LER, long-term energy restriction; Lox, lysyl oxidase; MHC, major histocompatibility complex; Mmp, matrix metalloproteinases; SER, short-term energy restriction; Tgf, transforming growth factor; WAT, white adipose tissue.

Manuscript received 14 July 2005. Initial review completed 26 August 2005. Revision accepted 3 November 2005.

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