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Adipose tissue energy metabolism: altered gene expression profile of mice subjected to long-term caloric restriction¹

YOSHIKAZU HIGAMI^*,, THOMAS D. PUGH^*,, GRIER P. PAGE, DAVID B. ALLISON, TOMAS A. PROLLA^|| and RICHARD WEINDRUCH^*,,,2

^* Wisconsin Primate Research Center, Madison, Wisconsin, USA;
Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA;
Geriatric Research, Education and Clinical Center; Veterans Administration Hospital, Madison, Wisconsin, USA;
Department of Biostatistics, Section on Statistical Genetics and Clinical Nutrition Research Center, University of Alabama, Birmingham, Alabama, USA; and
^|| Department of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin, USA

²Correspondence: Veterans Administration Hospital, 2500 Overlook Terrace, Madison, WI 53705, USA. E-mail: rhweindr{at}wisc.edu

SPECIFIC AIMS

Over the last decade, white adipose tissue (WAT) has transformed from "metabolic inertness" to being a source of multiple systemic metabolic regulators including those related to obesity-associated metabolic abnormalities. We investigated the influences of short-term and lifespan-prolonging long-term caloric restriction (LCR) on the expression of over 11,000 genes using oligonucleotide microarrays in four groups of 10- to 11-month-old male C57Bl6 mice: fasted for 18 h prior to death (F), subjected to short-term CR for 23 days (SCR), LCR for 9 months, and nonfasted control (CO) mice.

PRINCIPAL FINDINGS

1. Of the more than 11,000 genes studied, 6266 genes were determined to be expressed in WAT based on the Affymetrix algorithm. Among these, F, SCR and LCR altered the expression of 2, 2, and 345 genes, respectively, compared with CO based on meeting these three criteria for differential expression: bootstrap test (P<0.01), fold change (FC, exceeding up-regulations or down-regulations of 1.5-fold) and false discovery rate (FDR, <10%). These data suggest that 1) short-term CR minimally affects WAT gene expression profiles, 2) the metabolic adaptation during food shortage for the short term might be regulated post-transcriptionally in WAT; and 3) in contrast, LCR markedly influenced the gene expression profile.

2. Among 345 genes altered in expression level by LCR, 120 genes were associated with metabolism including carbohydrate, lipid, amino acid and central aspects of energy metabolism. Importantly, 86% of these (103 of 120 genes) were up-regulated by LCR (Table 1 ). Another 108 genes were classified as involved in either cytoskeleton, extracellular matrix, inflammation and angiogenesis. Nearly all of these (104 of 109 genes) were down-regulated by LCR (Table 1) . LCR up-regulated several genes previously demonstrated to characterize adipocyte differentiation.

3. LCR down-regulated the expression of most genes associated with cholesterol metabolism; however, LCR up-regulated the expression of most genes involved in glucose metabolism (glucose transporters, glycolysis, pentose phosphatase pathway, glycogen synthesis and gluconeogenesis), amino acid metabolism and lipogenesis (fatty acid transporter, ATP citrate lyase, fatty acid synthesis, and triacylglycerol synthesis, Table 1 ). We found that all 42 differentially expressed genes associated with mitochondrial energy metabolism were up-regulated by LCR, including genes encoding proteins involved in the Krebs cycle, fatty acid transport to mitochondria, mitochondrial ß-oxidation, electron transport, and oxidative phosphorylation (complexes I, II, III, IV, ATP synthase; Table 2 which lists 37 of the 42 upregulated genes involved in mitochondrial energy metabolism). Also induced by LCR was uncoupling protein 3 (twofold).

4. High cytochrome c oxidase activity was detected histochemically in a larger proportion of adipocytes of LCR mice compared with CO mice. Therefore, it is likely that LCR activates glucose, amino acid metabolism and mitochondrial energy metabolism and promotes lipogenesis, whereas cholesterol metabolism is down-regulated (Fig. 1 ).

View larger version (113K): [in this window] [in a new window]   Figure 1. Schematic diagram of WAT energy metabolism in mice subjected to LCR. The genes encoding transporters of glucose, amino acids and fatty acids were up-regulated by. LCR as was the expression of genes involved in glycolysis, portions of the lipolytic pathway, amino acid metabolism and mitochondrial energy metabolism including Krebs cycle, ß-oxidation, electron transport and oxidative phosphorylation, suggesting that the synthesis of ATP and NADPH + H+ are enhanced in WAT by LCR. LCR, increased the expression of genes involved in gluconeogenesis, glycogen synthesis and lipogenesis. Black boxes indicate genes or pathways up-regulated by LCR. Solid red lines show probable enhanced metabolic fluxes based on the gene expression profile. Dotted black lines indicate reduced metabolic fluxes (CRAT: carnitine acetyltransferase, CACT: carnitine/acylcarnitine translocase, CPT2; carnitine palmitoyltransferase 2).

5. Quantitative RT-PCR suggested that LCR down-regulated the expression of leptin and up-regulated SREBP1/ADD1 expression. It is possible that LCR-associated metabolic shifts in WAT may be explained, in part, by altered expressions of these two regulatory genes.

6. Histologically, the adipocytes of LCR mice appear smaller than those of CO mice. Multilocular adipocytes, which might represent an intermediate phenotype between white and brown adipocytes, were distinctly, although infrequently, observed in WAT of LCR mice but not in CO mice. There is evidence suggesting that the conversion from white to brown adipocytes might involve ß 3-adrenergic signaling. We observed that ß 3-adrenergic receptor mRNA was abundantly expressed and markedly up-regulated in WAT of LCR mice. Therefore, the multilocular adipocytes observed in LCR mice might result from ß 3-adrenergic signaling activated by LCR.

CONCLUSIONS AND SIGNIFICANCE

LCR remains the most robust, reproducible and simplest experimental manipulation known to extend maximum lifespan in many species and to retard a broad spectrum of age-associated pathophysiological changes in laboratory rodents. There is growing interest in discovering underlying mechanisms. The attenuation of oxidative and other stresses and modulation of glycemia and insulinemia may be significant factors in the actions of LCR, but the exact underlying mechanisms remain debatable.

Aging is associated with obesity, insulin resistance and leptin resistance, and all of these are opposed by LCR. Recently, several WAT-derived secretary molecules (adipocytokines) such as leptin, have been characterized and some of these are importantly involved in the metabolic abnormalities associated with obesity. Surgical removal of visceral fat prevents the obesity-induced and the age-associated insulin resistance in rats, possibly due to reduced synthesis of adipocytokines. Therefore, the protective action against the age-associated insulin and leptin resistance by LCR could result from the decreasing adiposity, and it is likely that WAT plays some role in aging and life span extension by LCR.

We observed major shifts in WAT gene expression caused by LCR, but not by F and SCR. Our data clearly show that, at the level of gene expression, LCR alters the characteristics of WAT metabolism, particularly mitochondrial energy metabolism. The gene expression data were supported by both RT-PCR and histological studies. It is likely that LCR-induced metabolic shifts in WAT play an important role in its beneficial actions (in addition to its reduction of adiposity). In the present study, we provide evidence that LCR shifts the transcriptional phenotype of WAT toward energy metabolism activation. Further, many of the shifts in gene expression following LCR are known to occur during adipocyte differentiation. These data provide new insights on the metabolic state associated with aging retardation in mice on LCR.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0678fje;

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