During the last century, humans have reached the longest life expectancy in history. However, it is remarkable that the increase in life expectancy is associated with a plethora of age-related pathologies, and it is estimated that only ~45% of humans reaching 75 years of age describe a good quality of life. These data indicate the need to develop novel effective therapies to prevent and treat age-related complications in order to promote healthy aging. From a biological point of view, aging is the consequence of the accumulation of a great variety of molecular and cellular modifications over time, which leads to a gradual decrease in physical, metabolic, and mental capacities, as well as an increased risk of disease and death. In mammals, most of the main age-related pathologies that shorten health and life expectancy, such as atherosclerosis, diabetes mellitus, sarcopenia, and hepatic steatosis, can be derived from improper metabolic control.
Acetyl-coenzyme A (Ac-CoA) is a central metabolite produced from all energy sources, such as amino acids, fatty acids, and carbohydrates. This small molecule plays a key role in a great number of essential cellular processes, including the endogenous synthesis of fatty acids, cholesterol, and coenzyme Q. Moreover, Ac-CoA is the universal acetyl group donor for protein acetylation, a post-translational modification that controls protein stability, localization, and function. ATP-citrate lyase (Acly) is the enzyme catalyzing the generation of nuclear-cytosolic Ac-CoA and oxaloacetate from citrate in the presence of ATP and coenzyme A. It is expressed ubiquitously, although greater expression is found in lipogenic tissues. Moreover, several cancers have been shown to exhibit aberrantly increased Acly activity. Acly is an essential enzyme, as demonstrated by the lack of viability of homozygous Acly knockout mice. However, heterozygous Acly knockout mice are healthy and fertile and exhibit normal lipid metabolism. These results have supported that a partial loss of Acly activity is compatible with an optimal quality of life.
The central role of Acly in de novo lipogenesis has fostered a need to generate therapeutic strategies based on the use of pharmacological inhibitors as a hypolipidemic strategy for metabolic syndrome and cancer treatment. Studies using short-term administrations of Acly inhibitors have reported promising results in refraining tumor growth or ameliorating metabolic parameters in mammals. Remarkably, research using bempedoic acid, a dual Ampk activator/Acly inhibitor developed for the treatment of dyslipidemia and cardio-metabolic disease, has provided positive results in lowering low-density lipoprotein cholesterol in clinical trials, and it is currently in the market. However, the mechanisms that govern the handling of sustained Acly inhibition, and particularly how such mechanisms orchestrate long-term cellular reprogramming, remain to be elucidated.
Here we assessed the consequences of long-term exposure to the Acly inhibitor SB-204990 in mice. We performed an unbiased multiomic approach integrating transcriptomics, proteomics, and untargeted metabolomics in murine hepatic tissue, given its central role in the maintenance of metabolic homeostasis. Analyses uncovered effects on energy metabolism, mitochondrial function, lipid metabolism, mTOR activity, as well as in the control of the folate cycle. Epigenetic studies indicated that these effects are not associated with global modulations in histone and non-histone protein acetylation. SB-204990 recapitulates certain effects of mTOR inhibitors in standard (STD)-fed mice and produces favorable effects in mice fed with a high-fat diet (HFD), which might provide therapeutic benefits against the current pandemic proportions of obesity-related metabolic disorders that predispose to unhealthy aging.
To define age-dependent metabolic changes that contribute to promote aging processes as well as to cause premature death, we evaluated different parameters of glucose homeostasis at different ages in healthy STD-fed wild-type mice. Body weight and the weight of several tissues were greater in adult and old mice (Fig. 1a, b). The assessment of oral glucose tolerance indicated the absence of major age-dependent alterations in glucose or insulin levels during the tests among young and old mice. However, significant differences, specifically in adult vs. old mice, in circulating insulin levels were observed (Fig. 1c–f). The analysis of pyruvate tolerance and insulin sensitivity indicated a reduced ability to promote hepatic gluconeogenesis as well as severe insulin resistance in aged mice (Fig. 1g–j). In fasting conditions, old mice exhibited hyperinsulinemia while maintaining normoglycemia, producing a greater index of the homeostatic model assessment of insulin resistance (HOMA-IR) (Fig. 1k–m). These data indicated that the most prominent effects of aging involve a deterioration of functionality in insulin-target tissues in the control of glucose metabolism, suggesting that these effects could contribute to the aging phenotype. We then focused on determining whether the expression levels of Acly, a gene that occupies a central role in the carbohydrate-lipid metabolism interface, are altered in an age-dependent manner in metabolic tissues. Remarkably, old mice exhibited higher levels of Acly expression and enzyme activity in the liver (Fig. 1n and Supplementary Fig. S1a). These data suggest that hepatic Acly could play an important role in the development of metabolic alterations that occur during the aging process.
Fig. 1: Hepatic Acly expression is increased in aging mice.
a Body weight. n = 4. One-way ANOVA. b Tissue weight. n = 4. One-way ANOVA. c OGTT. n = 8. Two-way ANOVA repeated measures. d Area under the curve (AUC) of the OGTT. n = 8. One-way ANOVA. e Insulin levels during an OGTT. n = 8. Two-way ANOVA repeated measures. f AUC of insulin levels during an OGTT. n = 8. One-way ANOVA. g IPPTT. n = 8 for young, n = 8 for adult, n = 7 for old. Two-way ANOVA repeated measures. h AUC of the IPPTT. n = 8 for young, n = 8 for adult, n = 7 for old. One-way ANOVA. i Insulin tolerance test (ITT). n = 7 for young, n = 7 for adult, n = 6 for old. Two-way ANOVA repeated measures. j AUC of the ITT. n = 7 for young, n = 7 for adult, n = 6 for old. One-way ANOVA. k Circulating glucose levels at 16 h of fasting. n = 8. One-way ANOVA. l Circulating insulin levels at 16 h of fasting. n = 8 for young, n = 6 for adult, n = 8 for old. One-way ANOVA. m HOMA-IR index. n = 8 for young, n = 6 for adult, n = 8 for old. One-way ANOVA. n Acly gene expression. n = 3–4. a.u.: arbitrary units. r.u.: relative units. BAT: Brown adipose tissue. Gastroc: Gastrocnemius. Ud: Under the threshold of detection.Data shown are the means ± SEM. *p< 0.05 Old vs. Young; #p< 0.05 Adult vs. Old; &p< 0.05 Young vs. Adult.
In physiological conditions, Acly has been proposed as the main producer of cytosolic Ac-CoA. Cytosolic Ac-CoA is used for endogenous lipid production and for the production of malonyl-coenzyme A, an inhibitor of the carnitine palmitoyltransferase I, required for fatty acid uptake into the mitochondria. Radiolabeled [H3]-glucose incorporation into lipids was measured to indirectly assess liponeogenesis in primary hepatocytes isolated from male wild-type mice. Results indicated that SB-204990 elicits a dose-dependent inhibition of glucose-dependent de novo lipogenesis (Supplementary Fig. S2a). Cell death measured via ELISA and urea secretion indicated that toxicity occurs at concentrations greater than 10 µM, with marked toxicity at 100 µM (Supplementary Fig. S2b
