Deficiency of the lipid synthesis enzyme, DGAT1, extends longevity in mice.

Calorie restriction results in leanness, which is linked to metabolic conditions that favor longevity. We show here that deficiency of the triglyceride synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which promotes leanness, also extends longevity without limiting food intake. Female DGAT1-deficient mice were protected from age-related increases in body fat, tissue triglycerides, and inflammation in white adipose tissue. This protection was accompanied by increased mean and maximal life spans of ~25% and ~10%, respectively. Middle-agedDgat1-/- mice exhibited several features associated with longevity, including decreased levels of circulating insulin growth factor 1 (IGF1) and reduced fecundity. Thus, deletion of DGAT1 in mice provides a model of leanness and extended lifespan that is independent of calorie restriction.


INTRODUCTION
The amount of fat mass of an organism is emerging as key determinant in longevity. Too little or too much fat is associated with early mortality in rodents and humans, whereas leanness, intermediate with respect to these two extremes is associated with relative longevity, possibly reflecting an optimal amount of fat. The most effective intervention to promote leanness and increase lifespan is calorie restriction (CR) [1]. CR, with adequate nutrient intake, extends the lifespan of yeast, invertebrates (worm and fly) and mice [2][3][4], and is associated with favorable changes in energy metabolism [5]. However, CR requires markedly restricting food intake, which stimulates appetite and induces hunger, making CR difficult to maintain.
An alternative strategy for leanness is to limit the accumulation of body fat by activating energy expendi-

Research Paper
ture. Although many interventions promote energy expenditure, we focus here on inhibiting acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which catalyzes the synthesis of triglycerides (TG) [6,7]. We showed previously that male DGAT1-deficient (Dgat1 -/-) mice have reduced adiposity associated with increased energy expenditure and normal-to-increased food intake [8,9]. These mice also exhibit delayed intestinal fat absorption [10] and favorable metabolic changes, including enhanced insulin and leptin sensitivity [11,12], resistance to diet-induced obesity, tissue steatosis, and glucose intolerance [8,13]. Given these beneficial metabolic phenotypes, we hypothesized that DGAT1 deficiency protects against the metabolic consequences of aging and extends longevity. In this study, we examined lifespan and age-related changes in metabolism in female wild-type (WT) and Dgat1 -/mice.

Dgat1 -/mice are protected against age-related metabolic changes
We compared energy balance in young and middle-aged female WT and Dgat1 -/mice fed a chow diet (fat content 10%). Food intake [g/g lean body mass (LBM)/24h] was similar in young (3-4 month-old) and middle-aged (12-15 month-old) groups of WT and Dgat1 -/mice, though it trended higher in Dgat1 -/mice ( Figure 1A). Oxygen consumption (ml/g LBM/h; average 48 h), a measure of energy expenditure, was similar in young mice of either genotype, but was higher in middle-aged Dgat1 -/mice ( Figure 1B). This increase was present during both the light and dark cycles (data not shown). Although young WT and Dgat1 -/mice had similar body weights ( Figure 1C), the greater energy expenditure in Dgat1 -/mice was associated with ~10% less body weight by 9 months of age that is sustained through middle age ( Figure 1C).
Although bone mineral density (BMD), bone mineral content (BMC), and fat mass were similar in young mice of either genotype (Supplemental Figures 1A and  B, and Figure 1D), lean mass was higher in young Dgat1 -/mice ( Figure 1D). In middle-aged WT mice, BMD, BMC (Supplemental Figures 1A and B) and lean and fat masses were increased compared to young WT mice ( Figure 1D). In contrast, middle-aged Dgat1 -/mice showed no increases in BMD, BMC (Supplemental Figures 1A and B) or lean mass and had smaller increases in fat mass ( Figure 1D). In agreement with the lower fat mass and bone density, serum leptin levels were lower in middle-aged Dgat1 -/mice than in WT controls (Supplemental Figure 1C). -/mice (n = 11-12/genotype). (C) Reduced body weight and (D) fat mass in Dgat1 -/mice. Body weight was measured monthly in mice from the aging cohort (n = 30/genotype). Total body mass is expressed as fat and lean mass composition for young and middle-aged groups (n = 11-12/genotype). "Young" and "Middle-aged" refer to ages 3-4 months (mo) and 12-13 mo, respectively (*p < 0.05 vs. WT. # p <0.05 vs. same genotype, different age). www.impactaging.com The reduced fat mass of Dgat1 -/mice was not confined to white adipose tissue (WAT), as middle-aged Dgat1 -/mice also exhibited a ~40-50% lower levels of triglyceride (TG) in heart and skeletal muscle ( Figure  1E). Liver TG content also trended lower in middleaged Dgat1 -/mice (~20%, p = 0.059, data not shown). TG accumulation in tissues can be associated with tissue inflammation, which has been identified as a signature of aging [14][15][16]. Indeed, despite the relatively low baseline of inflammation in mice fed a chow diet, the reduced adiposity seen in Dgat1 -/mice was associated with fewer total leukocytes in inguinal fat ( Figure 1F). This decrease was due, at least in part, to a reduction in the number of recruited and resident macrophages (F4/80-positive cells; Figure 1G). Of note, the weights of inguinal fat pads were essentially undistinguishable between the groups, consistent with our previous findings in non-pregnant Dgat1 -/females [17]. Together, these data show that increased energy expenditure and reduced adiposity in middle-aged Dgat1 -/mice is associated with less inflammation in the WAT.

Female Dgat1 -/mice have an extended lifespan
We next determined if the leanness and reduction in age-related metabolic consequences in Dgat1 -/miceaffected their longevity. Remarkably, the average lifespan in female Dgat1 -/mice was ~5-6 months (~25%) longer than that for WT mice ( Figure 2 and Table 1). The maximal and minimal lifespan (calculated as the mean of the oldest or youngest 10% of mice within a genotype) of Dgat1 -/mice were also extended by ~3 and 6 months, respectively (Table 1; p < 0.0001).
The extended longevity of female Dgat1 -/mice was accompanied by factors that correlate with lifespan extension. Diminished insulin-like growth factor-1 (IGF-1)/insulin signaling can promote longevity in worms, flies and mice [5, 18-21]. Serum IGF-1 levels were similar in young WT and Dgat1 -/mice, but were ~30% lower in middle-aged Dgat1 -/mice than in WT controls ( Figure 3A). Although fasting levels of blood glucose trended higher in young Dgat1 -/mice and were significantly higher with age than in WT controls (data not shown), serum insulin levels ( Figure 3B) and glucose tolerance (Supplemental Figure 2A) were similar in both groups of middle-aged Dgat1 -/mice. This likely suggests that there were not large differences in insulin sensitivity. These findings in middle-aged, female mice contrast with those of young, male Dgat1 -/mice [11], which are more insulin-sensitive, suggestive of a possible sexual dimorphism for this aspect of the DGAT1 deficiency phenotype.
Reduced fecundity also correlates with lifespan extension [22,23]. We observed that Dgat1 -/females had an average litter size of 3.8 pups compared with 7.4 pups for WT ( Figure 3C). This observation is consistent with previous studies demonstrating an inverse relationship between reproduction and longevity [24].

Figure 2. Extended longevity in female Dgat1 -/mice. Survival curves for female wild-type
and Dgat1 -/mice (n=30 per genotype). Further analysis of the data is summarized in Table 1. www.impactaging.com

DGAT1 deficiency mimics some but not all aspects of calorie restriction
The finding that DGAT1 deficiency, like CR, extends longevity in mice prompted us to perform a more detailed phenotypic comparison of DGAT1-deficient mice with those subjected to CR. Beyond lifespan extension, many aspects of Dgat1 -/mice (e.g., reduced adiposity, non-adipose tissue TG, tissue inflammation, bone density, fecundity, and decreased serum leptin and IGF1 levels) are similarly seen in CR [1,5,15,19,23,25]. However, there were also notable physiological differences. First, Dgat1 -/mice ate as much or more than WT mice ( Figure 1A). In addition, hepatic mitochondrial biogenesis and function are increased during CR [26], possibly to reduce oxidative stress, but we found that mitochondrial DNA and citrate synthase activity were similar in the livers of middle-aged WT and Dgat1 -/mice (Supplemental Figure 2B and C).
To further investigate the changes in response to CR and DGAT1 deficiency, we performed whole-genome microarray and pathway analyses on liver samples of WT mice fed a calorie restricted diet and Dgat1 -/mice fed ad libitum. Middle-aged female WT mice were subjected to short-term CR (WTCR), in which intake was restricted by 25% for 2 weeks and 50% for the following 2 weeks (achieving ~3 g of weight loss every 2 weeks), while female middle-aged Dgat1 -/mice were fed ad libitum (Dgat1 -/-AL). To identify genes whose mRNA levels were significantly altered, both groups were compared with WT controls fed ad libitum (WTAL).  www.impactaging.com www.impactaging.com A subset of genes with altered mRNA levels was validated by quantitative RT-PCR (Supplemental Figure  3). Among these validated genes, we found that DGAT1 expression was up-regulated ~twofold in response to CR. Overall, nearly seven times as many genes were altered with CR than in Dgat1 -/-AL (Figure 4 and Supplemental Table 1), suggesting that CR and DGAT1 deficiency alter physiology and promote lifespan extension in different ways. Still, we discovered a relatively small subset of about 100 commonly up-or down-regulated genes (Figure 4, Supplemental Table 1). These genes are of potential interest since they might point to common pathways that promote murine longevity. Notably, both CR and DGAT1 deficiency caused significant down-regulation of immune/inflammation response and cholesterol biosynthesis pathways (Figure 4, clusters C4 and C5). Although several genes involved in inflammation and immune responses were similarly regulated (Emr1, Ccl5, C1qa and Ccl3; Supplemental Table 1), the most highly down-regulated transcripts were Fabp5, Igj and Cxcl13 (Supplemental Table 1). These findings are consistent with the decreased levels of markers of inflammation in WAT of middle-aged Dgat1 -/mice ( Figure 1F and G). Both WTCR and Dgat1 -/-AL mice showed down-regulation of genes involved in the cholesterol biosynthesis pathway (Fdft1, Fdps, Hmgcs1, Mvd, and Sqle; Supplemental Figure 4), a finding that was also observed in the livers of long-lived Snell dwarf (Pit1 dw/dwJ ) and ribosomal protein S6 kinase 1 knockout mice (S6K1 -/-) mice [27, 28]. Furthermore, circulating levels of total cholesterol were ~35% lower in middleaged female Dgat1 -/mice than WT controls (39 ± 3 vs. 61 ± 5 mg/dL in WT; p < 0.01). Of note, DGAT1 deficiency in apolipoprotein E-deficient mice decreases cholesterol absorption, blood cholesterol, and foam cell formation, resulting in protection from atherosclerosis [29]. Thus, DGAT1 deficiency, like CR, may promote favorable changes in lipid metabolism that promote extended lifespan.

Summary and Implications
Our findings show that deletion of the TG synthesis enzyme, DGAT1, promotes leanness and extends lifespan in female mice and therefore suggests a link between murine lipid metabolism and longevity. These results are consistent with a study linking lipid metabolism and longevity in Caenorhabditis elegans, where activation of lipid hydrolysis resulted in decreased fat mass and extended lifespan [30]. We presume that the effects of DGAT1 deficiency are the result of reduced TG and related lipid metabolites in tissues. However, notably, DGAT1 has several biochemical activities [6], and we therefore cannot exclude the possibility that changes in other DGAT1associated pathways contribute to these effects.
We propose that DGAT1 deficiency, by increasing energy expenditure and maintaining leanness, activates a metabolic program favorable for extending lifespan without restricting calories. Although the details of this process remain unclear, both DGAT1 deficiency and CR alter parameters related to cholesterol metabolism and inflammation, two processes linked to atherosclerosis and cardiovascular disease. The phenotype of Dgat1 -/mice is similar to that of mice lacking insulin receptors in WAT, which are also lean, have increased energy expenditure and food consumption and exhibit extended longevity [31,32]. The physiological alterations in both of these models may limit the accumulation of body fat and protect from the adverse metabolic consequences of excessive fat storage. Our findings suggest that inhibition of DGAT1, or other strategies to promote leanness, may have the potential to retard age-related metabolic disease and prolong lifespan in humans.

METHODS
Mice. and placed on ice for 20 min. Protein K (14 mg/ml) and RNase (10 mg/ml) were added to each sample and incubated overnight at 50°C. Mitochondrial DNA was then extracted using an equal volume of phenol/chloroform and 1/5 volume of 5 mM NaCl, and precipitated in an equal volume of isopropanol at -20°C overnight. After centrifugation at 12,000g at room temperature, the resulting pellet of mtDNA was washed with 70% ethanol and then dried. The pellet was resuspended in 10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA and 20 g/ml RNase. Genomic DNA content did not differ among groups.