Bruss MD, Khambatta CF, Ruby MA, Aggarwal I, Hellerstein MK. Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates. Am J Physiol Endocrinol Metab 298: E108-E116, 2010. First published November 3, 2009 zdoi:10.1152/ajpendo.00524.2009.-Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However, at metabolic steady state, FA oxidation must match FA intake plus synthesis; moreover, FA intake is low, not high, during CR. Therefore, it is not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole body fuel selection, endogenous FA synthesis, and gene expression in mice on CR. Within 2 days of CR being started, a shift to a cyclic, diurnal pattern of whole body FA metabolism occurred, with an initial phase of elevated endogenous FA synthesis [respiratory exchange ratio (RER) Ͼ1.10, lasting 4 -6 h after food provision], followed by a prolonged phase of FA oxidation (RER ϭ 0.70, lasting 18 -20 h). CR mice oxidized four times as much fat per day as ad libitum (AL)-fed controls (367 Ϯ 19 vs. 97 Ϯ 14 mg/day, P Ͻ O.001) despite reduced energy intake from fat. This increase in FA oxidation was balanced by a threefold increase in adipose tissue FA synthesis compared with AL. Expression of FA synthase and acetyl-CoA carboxylase mRNA were increased in adipose and liver in a timedependent manner. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for oxidative damage and disease risk. fat synthesis; lipogenesis; palmitoleate; heavy water CALORIE RESTRICTION (CR) delays the development of chronic disease and prolongs lifespan in mice (1,17,27,34). These effects correlate with a rapid induction in the expression of certain genes that persist as long as animals remain on CR (10, 36) even after energy balance is restored. These observations suggest the presence of a chronic signal of reduced energy availability that persists after energy balance has been reestablished. However, the underlying metabolic signals and adaptations responsible are not fully understood.Mice on CR regimens have been reported to exhibit increased expression of genes for fatty acid (FA) oxidation and decreased expression of genes for FA synthesis compared with ad libitum (AL)-fed controls (6,7,30,38). Due to differential entry points into the electron transport chain, a metabolic shift from carbohydrate to FA oxidation may reduce the production of reactive oxygen species (ROS) (15). A shift to FA oxidation thereby represents a potential mechanism for reduced oxidative damage, which has been proposed as a potential explanation for the health benefits of CR (14,15,29,35). It has also be...

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

SummaryCombating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age‐related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins in vivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie‐restricted (CR), and rapamycin (Rapa)‐treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half‐lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa‐treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S‐transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals.

Bruss MD, Thompson AC, Aggarwal I, Khambatta CF, Hellerstein MK. The effects of physiological adaptations to calorie restriction on global cell proliferation rates. Am J Physiol Endocrinol Metab 300: E735-E745, 2011. First published February 1, 2011 doi:10.1152/ajpendo.00661.2010 reduces the rate of cell proliferation in mitotic tissues. It has been suggested that this reduction in cell proliferation may mediate CR-induced increases in longevity. However, the mechanisms that lead to CRinduced reductions in cell proliferation rates remain unclear. To evaluate the CR-induced physiological adaptations that may mediate reductions in cell proliferation rates, we altered housing temperature and access to voluntary running wheels to determine the effects of food intake, energy expenditure, percent body fat, and body weight on proliferation rates of keratinocytes, liver cells, mammary epithelial cells, and splenic T-cells in C57BL/6 mice. We found that ϳ20% CR led to a reduction in cell proliferation rates in all cell types. However, lower cell proliferation rates were not observed with reductions in 1) food intake and energy expenditure in female mice housed at 27°C, 2) percent body fat in female mice provided running wheels, or 3) body weight in male mice provided running wheels compared with ad libitum-fed controls. In contrast, reductions in insulin-like growth factor I were associated with decreased cell proliferation rates. Taken together, these data suggest that CR-induced reductions in food intake, energy expenditure, percent body fat, and body weight do not account for the reductions in global cell proliferation rates observed in CR. In addition, these data are consistent with the hypothesis that reduced cell proliferation rates could be useful as a biomarker of interventions that increase longevity. food intake; energy expenditure; percent body fat; body weight CALORIE RESTRICTION (CR), defined as a reduction in caloric intake without malnutrition, increases lifespan, delays the onset of age-related diseases, and slows the functional decline of various organs (42). Although the mechanisms mediating these effects are unclear, one of the most rapid and robust effects of CR is a reduction in the rate of cell proliferation in mitotic tissues (25,28,38). The turnover rates of keratinocytes, liver cells, mammary epithelial cells (MECs), splenic T cells, and prostate cells are decreased by 30 -50% following several weeks of CR, and the effects persist throughout the intervention period (25). It has been suggested that this reduction in cell proliferation could mediate many of the longevity and anticancer effects of CR by delaying replicative senescence and reducing the promotional phase of carcinogenesis (11,42). Considering that the rate of cell proliferation is an integrated response that comprises many genetic and hormonal signals and appears to be mechanistically linked to aging, this cellular process represents a potential target for monitoring therapeutic interventions aimed at delaying the onset of age-relate...

The glucocorticoid (GC) receptor (GR) has multiple effector mechanisms, including dimerization-mediated transactivation of target genes via DNA binding and transcriptional repression mediated by protein-protein interactions. Much attention has been focused on developing selective GR modulators that would dissociate adverse effects from therapeutic anti-inflammatory effects. The GR(dim/dim) mouse has a mutation in the dimerization domain of GR and has been shown to have attenuated transactivation with intact repression. To understand the role of GR dimerization-dependent targets in multiple tissues, we measured metabolic fluxes through several disease-relevant GC target pathways using heavy water labeling and mass spectrometry in wild-type and GR(dim/dim) mice administered the potent GC dexamethasone (DEX). Absolute triglyceride synthesis was increased in both wild-type and GR(dim/dim) mice by DEX in the inguinal and epididymal fat depots. GR(dim/dim) mice showed an exaggerated response to DEX in both depots. De novo lipogenesis was also greatly increased in both depots in response to DEX in GR(dim/dim), but not wild-type mice. In contrast, the inhibitory effect of DEX on bone and skin collagen synthesis rates was greater in wild-type compared with GR(dim/dim) mice. Wild-type mice were more sensitive to DEX-dependent decreases in insulin sensitivity than GR(dim/dim) mice. Wild-type and GR(dim/dim) mice were equally sensitive to DEX-dependent decreases in muscle protein synthesis. Chronic elevation of GCs in GR(dim/dim) mice results in severe runting and lethality. In conclusion, some metabolic effects of GC treatment are exaggerated in adipose tissue of GR(dim/dim) mice, suggesting that selective GR modulators based on dissociating GR transactivation from repression should be evaluated carefully.