ES2965779A1 - ATP-citrate lyase inhibitors as geroprotective agents - Google Patents

Abstract

ATP-citrate lyase inhibitors for use as geroprotective agents, to treat metabolic disorders related to obesity and unhealthy aging.

Description

DESCRIPTION

ATP-citrate lyase inhibitors as geroprotective agents

FIELD OF TECHNIQUE

The invention is framed within the field of Medicine, more specifically within the framework of cellular reprogramming therapies that aim to molecularly modulate the mechanisms of aging, in order to locate compounds and/or treatments with therapeutic potential for the treatment of diseases linked to unhealthy aging such as fatty liver, diabetes and sarcopenia among others.

BACKGROUND

Over the past century, humans have achieved the longest lifespan in history. However, increased life expectancy is associated with a long list of age-related pathologies and only 45% of humans reach 75 years of age with a good quality of life. There is a clear need to develop new effective therapies to prevent and treat age-related complications in order to promote healthy aging.

Aging is the consequence of the accumulation of a wide variety of molecular and cellular modifications over time that leads to a gradual decline in physical, metabolic and mental capabilities, as well as an increased risk of disease and death. In mammals, most age-related pathologies, such as atherosclerosis, diabetes mellitus, sarcopenia and hepatic steatosis, can arise from inadequate metabolic control. In this scenario, finding a molecule that improves metabolic control and promotes healthy aging could have a major impact on humans worldwide.

Acetyl-coenzyme A (Ac-CoA) is an essential metabolite that can be produced from amino acids, fatty acids and carbohydrates. This small molecule plays a key role in a large number of essential cellular processes, including the endogenous synthesis of lipids including cholesterol, fatty acids and coenzyme Q. In addition, Ac-CoA is the universal donor of groups acetyl for protein acetylation, a post-translational modification that controls protein stability, localization, and function.

For its part, ATP-citrate lyase (ACLY) is the enzyme that catalyzes the generation of nuclear and cytosolic Ac-CoA, and oxaloacetate from citrate in the presence of ATP and coenzyme A.

ACLY is expressed ubiquitously, although it has a higher expression in lipogenic tissues (Chypreet al., 2012). ACLY is an essential enzyme, as demonstrated by the lack of viability of homozygous knockout mice for ACLY (Beigneuxet al., 2004). However, heterozygous ACLYknockout 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 (Beigneuxet al., 2004).

The central role of ATP-citrate lyase (ACLY) in denovolipogenesis has promoted the need to generate therapeutic strategies based on the use of pharmacological inhibitors as a lipid-lowering strategy for metabolic syndrome and cancer treatment (Granchi, 2018). The use of ACLY inhibitors has shown important effects in reducing circulating cholesterol levels. Studies using short-term administrations of ACLY inhibitors have shown promising results in slowing tumor growth or improving metabolic parameters in mammals (Pinkoskyet al.,2013). In this sense, research with bempedoic acid developed for the treatment of dyslipidemia and cardiometabolic disease, with a dual effect: activator of Ampk (AMP-activated protein kinase) and inhibitor of ACLY (Pinkoskyet al., 2013), has provided positive results in lowering low-density lipoprotein cholesterol in clinical trials and is currently on the market. However, the physiological consequences, as well as the molecular mechanisms that operate in the response to pharmacologically induced long-term inhibition of ACLY, and therefore the reduction of Ac-CoA levels, are unknown. A reduction in Ac-CoA levels can be expected to lead, in principle, to the inhibition of endogenous synthesis of fatty acids and cholesterol and the promotion of the deacetylated state of "acetylatable" proteins.

BRIEF SUMMARY OF THE INVENTION

The present invention proposes the inhibition of ACLY in order to treat metabolic disorders related to obesity and unhealthy aging.

In the present invention, the physiological consequences and altered molecular mechanisms have been evaluated in different species (yeasts, flies and mice) treated with the ACLY inhibitor SB-204990 (commercially available) and it has been shown that it significantly extends life in yeast and in flies, increasing markers of quality of life in flies, thus demonstrating its potential as a geroprotective agent.

Herein the concept of "geroprotective agent" or "geroprotective substance" is defined as a therapeutic agent that inhibits aging and age-related diseases or disorders, and prolongs the life of the individuals to whom it is supplied. The geroprotective effect is characterized by the improvement of metabolic control and the promotion of healthy aging, as well as the prevention of premature aging. Geroprotective compounds or substances have effects on the origin of aging, intervening in the metabolism of individuals. In our case, the intervention in the metabolism of yeast, flies and mice occurs through the inhibition of ACLY, which, as shown later in the memory and examples, has direct effects on the prevention of age-related diseases and delays aging, and finally, the death of individuals. The mechanisms of action involved are described in the report as well as the interconnected cellular pathways that contribute to the maintenance of metabolic health and healthy aging.

To determine whether long-term ACLY inhibition can provide metabolic health and quality of life benefits in mammals, long-term experiments (15 weeks) have been performed in wild-type mice treated with SB-204990. The mice have been fed either a standard healthy diet (STD) or a high-fat diet (HFD) that promotes unhealthy aging and premature death.

It should be taken into account that the administration of a high-fat diet (HFD) to the mice object of this study is intended to suddenly provoke the alterations for which ACLY inhibition is proposed to be treated. Due to the restrictions inherent to working with mice, we start from healthy populations that are provoked in a very short period of time, through the diet supplied, to metabolic diseases, alterations in functional capacity and neurocognitive health. Throughout the memory, and especially with reference to the embodiment examples, it must be understood that when we refer to individuals with high-fat diets we are referring to individuals who present metabolic alterations, in their functional capacity and in their neurocognitive health, all caused by the provision of a high-fat diet.

These three aspects, metabolic, functional and cognitive, account for the overall health of the individual, and their alterations are the object of treatment of the present invention.

The geroprotective effect of ACLY inhibitors, which is widely demonstrated throughout the examples of the invention, translates into their ability to restore normal values in the parameters that determine each of these aspects of the individual's global health.

Thus, when in the examples of memory realization we talk about individuals subjected to high-fat diets, it must be understood that we are referring to individuals in whom, through the diet provided, alternations have been provoked:

- metabolic; which are reflected in measurable parameters such as blood levels of glucose, insulin, pyruvate, cholesterol, HD grade acids, among others.

- in its functional capacity; which are reflected in measurable parameters such as strength, endurance and motor coordination.

- in your neurocognitive health; with special attention to memory.

Therefore, the scope of the invention should not be limited to individuals who have high-fat diets, but rather refers to individuals with pathological levels or with values outside of those considered healthy in each of the parameters that will be subject to assessment throughout. throughout the examples and which are briefly mentioned here.

Likewise, it is worth noting that many of these metabolic, functional and neurocognitive alterations, in non-experimental conditions, may be due not only to a high-fat diet or other unhealthy health habits, but also appear associated with aging. Initially healthy individuals can develop these alterations due to the aging process, regardless of their diet. Likewise, there are multiple other factors that can trigger the metabolic, functional and neurocognitive alterations that are the subject of treatment with the ACLY inhibitor, so the invention is not restricted to any specific triggering factor.

Thus, the patients who can potentially be treated to counteract the different metabolic, functional, and neurocognitive alterations indicated in the present invention will be defined by the presence of levels considered pathological, with values outside of healthy ones, or at risk, for any of the following parameters: glucose level, pyruvate level, insulin level, low and very low density lipoprotein level, cholesterol level and lipid content in liver tissue.

The levels that are considered pathological, out of normal or at risk for these parameters are not fixed values. These values are reviewed over time due to the development of medical practice; they vary depending on the methods, tests and measurement conditions, and also depending on the different population groups, sex, age, and other conditions. of individuals, as well as co-morbidities or added risk factors. Therefore, the concepts of "normal values" and "pathological values" must be understood in a broad sense, without constituting any limitation of the scope of application of the invention. In each case, the medical staff will determine if a specific individual has metabolic, functional or neurocognitive alterations based on the values obtained in the different assessment tests of these parameters and is therefore considered a candidate to be treated with ACLY inhibitors. of the present invention.

When we refer to metabolic alterations, we are also talking about diseases caused directly or indirectly by said metabolic alterations, such as, by way of example and not exhaustively, diabetes or fatty liver disease, as well as other conditions that may cause health risk factors, see, overweight, obesity, increased body weight, increased white adipose tissue mass or increased adipocyte size.

Throughout the examples of the invention, the effectiveness of long-term treatment with ACLY inhibitors is tested in all the parameters that have been the subject of study. The ACLY inhibitor used for the embodiments was SB-204990. Other ACLY inhibitors, equally effective for use within the framework of the present invention are: BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex and MEDICA16. All of them can be used to inhibit ACLY individually or in any of their possible combinations.

The studies carried out reveal that glucose tolerance and pyruvate tolerance improved in HFD-fed mice treated with the ACLY inhibitor SB-204990. The HOMA-IR index, a homeostatic model for assessing insulin resistance, was lower, and low- and very-low-density lipoproteins (LDL/VLDL), both atherogenic, were also lower, indicating an overall improvement in metabolic health. Therefore, SB-204990 provides therapeutic benefit against obesity-related metabolic disorders that predispose to unhealthy aging.

Interestingly, mice fed a healthy diet exhibited metabolic dysregulation with moderate insulin resistance, reproducing certain deleterious effects observed in mice treated with the geroprotective agent rapamycin.

To carry out this study, after 15 weeks of treatment with SB-204990, the mice were sacrificed to analyze their liver. In these analyses, it has been determined that key pathways that regulate aging in mice are altered in mice treated with the ACLY inhibitor. Thus, SB-204990 plays an important role in the regulation of mitochondrial function, while, surprisingly, no alterations in global histone and total protein acetylation are observed. This lack of difference in protein acetylation is explained by another of the physiological effects of SB-204990, a mild and sustained reduction in mitochondrial activity. Proof of this is that, both in primary cultures of hepatocytes treated with SB-209440 under different conditions (acetate, citrate, high glucose and metformin, among other treatments), and in the liver of mice treated with SB-204990, SB-204990 increases the concentration of some lipids and reduces mitochondrial metabolic activity. In both cases they show reduced levels of phosphorylated Ampk. SB-204990 by inhibiting ATP-citrate lyase (ACLY) reduces mitochondrial activity through inhibition of Ampk. It has been proven that the effects on increased lipid content and restricted metabolic activity are reduced if hepatocytes are simultaneously treated with SB-204990 and metformin or AICAR (both Ampk activators). These results support that ACLY inhibition acts on Ampk signaling to modulate mitochondrial performance.

A first aspect of the invention relates to an ATP-citrate lyase (ACLY) inhibitor, hereinafter "ACLY inhibitor of the invention", for use as a geroprotective agent in a patient population that presents altered, pathological, or outside those considered healthy, of one or more of the metabolic parameters selected from the list that consists of: glucose level, pyruvate level, insulin level, low and very low density lipoprotein level, cholesterol level and content of lipids in liver tissue.

In a preferred embodiment of the invention, the ACLY inhibitor of the invention is SB-204990, BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex, MEDICA16, or any combination thereof.

Another aspect of the invention relates to the ACLY inhibitor of the invention, for use in the treatment of metabolic diseases. In a preferred embodiment, that inhibitor is SB-204990, BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex, MEDICA16, or any combination thereof.

In a more preferred embodiment, the metabolic disease to be treated with the ACLY inhibitor of the invention affects glucose, pyruvate and/or insulin tolerance.

In another more preferred embodiment, the metabolic disease to be treated with the ACLY inhibitor of the invention affects blood levels of low and very low density lipoproteins and/or cholesterol.

In another more preferred embodiment, the metabolic disease to be treated with the ACLY inhibitor of the invention affects the lipid content in the liver tissue.

In another more preferred embodiment, the metabolic disease to be treated with the ACLY inhibitor of the invention occurs with an increase in body weight, an increase in the mass of white adipose tissue and/or an increase in the size of adipocytes.

In another more preferred embodiment, the metabolic disease to be treated with the ACLY inhibitor of the invention is selected from the list consisting of diabetes and fatty liver.

Another aspect of the invention relates to the ACLY inhibitor of the invention, for use in improving functional capacity. In a preferred embodiment, that inhibitor is SB-204990, BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex, MEDICA16, or any combination thereof.

In a more preferred embodiment, the improvement of functional capacity refers to the improvement in the results of tests that measure the strength, endurance and/or motor coordination of individuals.

Another aspect of the invention relates to the ACLY inhibitor of the invention, for use in improving neurocognitive health. In a preferred embodiment, that inhibitor is SB-204990, BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex, MEDICA16, or any combination thereof. In an even more preferred embodiment, the inhibitor of the invention is SB-204990.

In a more preferred embodiment, improvement in neurocognitive health refers to improvement in the results of tests that measure individuals' memory.

An even more preferred embodiment of the invention relates to the ACLY inhibitor of the invention for use in the joint treatment of two or more of the metabolic diseases, which affect functional capacity or which affect neurocognitive health, described previously.

Another aspect of the invention relates to a composition comprising the ACLY inhibitors of the invention, for use as a geroprotective agent.

In a preferred embodiment, the composition further comprises an Ampk activator.

In another more preferred embodiment, the Ampk activator is metformin and/or AICAR.

In another even more preferred embodiment, the composition is a pharmaceutical composition.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Pharmacological inhibition of ACLY using SB-204990: effects in mice, yeast and flies. In mice: (A) Body weight. (B) Fabric weight. (C) OGTT. (D) Area under the curve (AUC) of the OGTT. (E) Insulin levels during an OGTT. (F) AUC of insulin levels during an OGTT. (G) IPPTT. (H) AUC of the IPPTT. (I) ITT. (J) AUC of the ITT. (K) Circulating glucose levels after 16 hours of fasting. (L) Circulating insulin levels after 16 hours of fasting. (M) HOMA-IR index. (N) ACLY gene expression. In yeast. (O) Chronological lifespan of wild-type BY4741 yeast treated or not with 10 ^M SB-204990. In flies: (P) Kaplan-Meier survival curve of flies treated or not with SB-204990 10 ^M. (Q) Spontaneous activity during the longevity assay in flies treated or not with 10 ^M SB-204990.

SB: SB-204990. Ut: Untreated. a.u.: arbitrary units. r.u.: relative units. Data shown are means ± SEM. In panels A-N: * p< 0.05 Old vs. Youths; # p< 0.05 Adult vs Elderly; &p< 0.05 Young vs. Adult. In panels O-Q: * p< 0.05 SB vs Ut.

Figure 2: Health in mice and lifespan in yeast and flies. In mice: (A) Liver ACLY activity. In yeast: (B) Chronological lifespan of wild-type BY4741 yeast treated or not with 100 ^M SB-204990. In flies: (C) Kaplan-Meier survival curve of flies treated or not with 100 ^M SB-204990. (D) Kaplan-Meier survival curve of flies treated or not with 1000 ^M SB-204990. (E) Spontaneous activity during the longevity assay in flies in the SB-204990 100 ^M assay.

(F) Spontaneous activity during the longevity assay in flies in assay SB-204990 1000 |<j>M.

SB: SB-204990. Ut: untreated. Of note: raw yeast and fly data are shared with the main figure. Data shown are means ± SEM. * p< 0.05 SB vs Ut or Old vs Young.

Figure 3: Long-term treatment with SB-204990 in HFD-fed and STD-fed mice. (A) Pharmacokinetic evaluation of circulating levels of SB-204990 in mice exposed to oral administration (30 mg/kg body weight) of SB-204990. (B) Body weight throughout the study. (C) OGTT at week 13 of treatment. (D) AUC of the OGTT. (E) IPPTT at week 9 of treatment. (F) AUC of the IPPTT. (G) ITT at week 11 of treatment. (H) AUC of the ITT. (I) Insulin levels during an OGTT at week 10 of treatment. (J) AUC of insulin levels during an OGTT. (K) 24-hour fasting glucose levels at week 14 of treatment. (L) AUC of glucose levels during 24-hour fasting.

SB: SB-204990. STD: standard diet. HFD: high fat diet. a.u.: arbitrary units. The data shown are the means ± SEM.* p< 0.05 different times vs. time 0 or STD-SB vs. STD. # p < 0.05 HFD-SB vs HFD.

Figure 4: Effect on viability and functionality of SB-204990 in primary pancreatic islets and hepatocytes. (A-C) Primary hepatocytes were isolated and exposed to different concentrations of SB-204990 for 16 hours. (A) Determination of glucose incorporation into lipids. (B) Cell death analysis by ELISA. (C) Determination of urea production. (D-F) Primary pancreatic islets were isolated and exposed to different concentrations of SB-204990 for 16 hours. (D) Cell death analysis by ELISA. (E) Determination of metabolic activity by MTT assay. (F) Determination of glucose-stimulated insulin secretion (GSIS). (G) Hepatic ACLY activity. (H) Average daily energy intake during the course of the study. (I) Fecal lipid content at week 10 of treatment. (J) Fasting-induced energy intake in week 8 of treatment.

SB: SB-204990. STD: standard diet. HFD: high fat diet. Ut: untreated. Lg: low glucose. Hg: High glucose. Glu: glucose. r.u.: relative units. Data shown are means ± SEM. * p < 0.05 SB vs untreated or STD-SB vs STD.

Figure 5: Physical health in mice (A) Fasting circulating glucose levels at week 14 of treatment. (B) Fasting circulating insulin levels at week 14 of treatment. (C) HOMA-IR index at week 14 of treatment. (D) Percentage of glycated hemoglobin (HbA1c) in blood at week 15 of treatment. (E) Serum LDL/VLDL levels at week 15 of treatment. (F) Serum total cholesterol levels at week 15 of treatment. (G) Time to fall on an accelerated rotarod at week 8 of treatment. (H) Time to fall in the cable suspension test in week 10 of treatment. (I) Fabric weight. (J) Representative images of hematoxylin and eosin staining in various tissues. Scale 200 ^m. (K) Mean adipocyte area.

SB: SB-204990. STD: standard diet. HFD: high fat diet. BAT: Brown adipose tissue. a.u.: arbitrary units. The data shown are the means ± SEM.* p< 0.05 STD-SB vs. STD. # p < 0.05 HFD-SB vs HFD.

Figure 6: Neurocognitive function. Spatial memory was evaluated with the Barnes Maze in week 12-13 of treatment. (A) Latency to target on day 5. (B) Latency to target on day 12. (C) Errors to target on day 5. (D) Errors to target on day 12. (E) Total trials of target attempts on day 5. (F) Total target attempts on day 12.

SB: SB-204990. STD: standard diet. HFD: high fat diet. SKM: Skeletal muscle. Data shown are means ± SEM. * p< 0.05 STD-SB vs STD. # p < 0.05 HFD-SB vs HFD.

Figure 7: Hepatic levels of histone acetylation. (A) AML12 cells were cultured under standard conditions and treated with the indicated doses of acetate, citrate, and SB-204990 for 16 hours. The glucose concentration under basal conditions is 17 mM and when supplemented it is 42 mM. Acidic histone isolations were performed and histone acetylation levels were analyzed by Western blotting. Representative images of Western blots and Ponceau staining are shown. (B) Acidic histone isolations were performed and histone acetylation levels were analyzed by Western blotting in the livers of mice treated or not with SB-204990 for 15 weeks. Representative images of Western blots and Ponceau staining are shown. (C) Quantification of histone acetylation levels of various residues is depicted. (D) Representative Western blot images of protein lysine acetylation are shown. Molecular weight markers are depicted on the left. (E) Densitometric quantification of hepatic protein lysine acetylation by Western blot. (F) Total Ac-CoA levels in liver tissue.

SB: SB-204990. STD: standard diet. HFD: high fat diet. Ac-Lys: Acetylated lysine. r.u.: relative units. The data shown are the means ± SEM* p< 0.05 STD-SB vs. STD. # p < 0.05 HFD-SB vs HFD.

Figure 8: Quantification of histone acetylation in AML12 cells. AML12 cells were cultured under standard conditions and treated with the indicated doses of acetate, citrate, and SB-204990 for 16 hours. The glucose concentration under basal conditions is 17 mM and when supplemented it is 42 mM. (A) Quantification of histone acetylation levels by multi-residue Western blotting is depicted.

SB: SB-204990. r.u.: relative units. Data shown are means ± SEM. * p < 0.05 versus untreated cells under the same culture conditions. SB-209440 did not produce significant alterations in histone acetylation.

Figure 9: SB-204990 modulates the metabolism of fatty acids, cholesterol and phospholipids. (A) Hepatic cholesterol esters. Chol-P: cholesterol palmitate. (B) Immunoblots for Hmgcs and Gapdh of liver homogenates. (C) Densitometric analyzes of immunoblots shown in panel E. (D) Hepatic fatty acids. (E) Hepatic triglyceride species. P: palmitic acid. O: oleic acid. L: linoleic acid. S: Stearic acid. (F) Analysis of significantly altered hydrophobic metabolites by LC/MS. (G) Analysis of significantly altered hydrophilic metabolites by LC/MS.

SB: SB-204990. STD: standard diet. HFD: high fat diet. r.u.: relative units. Data shown are means ± SEM. * p< 0.05 STD-SB vs STD. # p < 0.05 HFD-SB vs. HFD.

Figure 10: SB-204990-mediated effects on mitochondrial function and lipid accumulation. (A-F) Primary hepatocytes were treated with SB-204990 under different experimental conditions. Unless otherwise indicated, hepatocytes were treated for 16 hours with 10 ^M SB-204990. (A) Representative images of oil red O stained primary hepatocytes treated with 10 ^M SB-204990. Scale bar 50 ^m. (B) Quantification of oil red O staining of primary hepatocytes treated with 10 ^M SB-204990. (C-D) Quantification of oil red O staining and determination of metabolic activity by the MTT test in primary hepatocytes treated with 10 ^M SB-204990 in the presence of the indicated doses of acetate or citrate. The glucose concentration under basal conditions is 5 mM and when supplemented it is 25 mM. (C) Oil red O stain. (D) MTT test. (E) Oxygen consumption rate in primary hepatocytes. (F) Extracellular acidification rate in primary hepatocytes. (G) Oxygen consumption rate of freshly isolated hepatic mitochondria from mice fed SB-204990. Liver samples were collected 3 hours after gavage. (H) Immunoblots for liver proteins from mice treated with SB-204990 for 15 weeks. Densitometric quantification is shown in Figure S7E. (I-K) Primary hepatocytes were treated with SB-204990 or not in the presence of metformin (500 ^M) for 16 hours. (I) Representative images of primary hepatocytes stained with oil red O. Scale bar 50 ^m. (J) Quantification of Oil red O staining. (K) Quantification of metabolic activity by MTT.

STD: standard diet. HFD: high fat diet. SB: SB-204990. NS: not significant. r.u.: relative units. Data shown are means ± SEM. Unless otherwise indicated * p< 0.05 SB vs. Ut or LG-SB vs. LG. &p< 0.05 HGacetate vs acetate.

Figure 11: Effects of SB-204990 on mitochondria and cellular stress. (A) Representative images of primary hepatocytes stained with oil red O treated or not with 10 ^M SB-204990 in the presence of acetate or citrate. Scale bar 50 ^m. (B) Primary hepatocytes were treated with SB-204990 or not in the presence of metformin (500 ^M) for 16 hours. Immunoblots for total and phosphorylated Ampk and densitometric quantifications were performed. (C) Densitometric quantification of liver proteins by Western blotting of blots shown in Figure 7H. (D) Primary hepatocytes were treated with SB-204990 or not in the presence of AICAR, rapamycin, MHY1485, tetrahydrofolate, or methotrexate for 16 hours. Quantification of oil red O staining and metabolic activity by the MTT test of primary hepatocytes treated with 10 ^M SB-204990 in the presence or absence of the indicated compounds. (E) Immunoblots for liver proteins from mice treated with SB-204990 for 15 weeks. (F) Densitometric quantification of liver proteins by Western blot.

SB: SB-204990. STD: standard diet. HFD: high fat diet. SB: SB-204990. Metf: Metformin. Rapa: rapamycin, THF: tetrahydrofolic acid, Metho: methotrexate. NS: Not significant. r.u.: relative units. The data shown are the means ± SEM.* p< 0.05 SB versus Ut, SB-control versus Ut-control or STD-SB versus STD. # p< 0.05 SB-Ut-Metf versus Ut-Metf or HFD-SB versus HFD.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Longevity assays in yeast and Drosophila

Longevity assays were performed in yeast and flies to determine whether ACLY inhibitors could promote healthier aging and prolong the lifespan of both. The results can be seen in Figure 1O-P and Figure 2B-D.

On the one hand, the chronological life of the BY4741 strain of Saccharomyces cerevisiae was studied. It was cultured in complete synthetic dextrose (SDC) medium containing 2% glucose with a 4-fold excess of tryptophan, leucine, uracil and histidine supplements. was measured as described Fabrizio and Longo, 2003; Mirisolaet al.,2014. Chronological shelf life was monitored in expired SDC medium by measuring colony forming units (CFU) every 48-72 hours. The number of CFU on day 3 was considered 100% initial survival. Yeast were treated or not treated with SB-204990 on day 0.

For the study of the chronological life of Drosophila, fly stock w1118 (#5905) from the Bloomingtin Drosophila Stock Center was used. Flies were maintained on standard sugar/yeast/agar food containing low melting point agar (OmniPur® Agarose, Low Melting, CAS 9012-36-6, Merck) at 25°C on a light/dark cycle of 12 hours with a constant humidity of 60%. Drosophila crosses laid eggs at 25°C, and adult experimental flies were allowed to emerge and mate for 1 day. Subsequently, adult flies were anesthetized with CO2 and males were selected for experiments. A density of 8-10 flies per vial was used for shelf life evaluation. Spontaneous activity of flies in each vial was recorded hourly using the Tracked.bio platform and the average of the 5-day activity was calculated. Manual counts of flies were also performed weekly to determine survival. Fresh food was exchanged weekly.

SB-204990 (10 ^M), an ACLY inhibitor promoted longer lifespan in yeast, similar to the ACLY inhibitor hydroxycitric acid (Baroniet al.,2020), and also in flies (Figure 1O-P). Furthermore, spontaneous activity, which reflects the health of flies, was significantly higher early in life (Figure 1Q). Higher concentrations of SB-204990 (100-1000 ^M) did not produce an extension of lifespan in yeast and flies, while spontaneous activity increased in the initial stages of survival assays (Figure 2B-F).

These data indicate that the ACLY inhibitor SB-204990 is efficient in extending lifespan beyond evolutionary limits.

Example 2: Tests with mice

Animals, diets and in vivo treatments

Experiments with mice were approved by the CABIMER Animal Committee and were carried out in accordance with the Spanish law on the use of animals RD 53/2013 and the EU Directive (2010/63/EU) for research with animals. 8-week-old wild-type male C57BL/6 mice were purchased. The mice were distributed into four different groups at 26 weeks of age and the treatments were started:

(1)STD: standard diet with rodent food TD2914 (Envigo, Barcelona, Spain) with the following percentages of calorie distribution: 67% carbohydrates, 20% proteins and 13% fats,

(2)STD-SB: STD 250 mg/Kg of SB-204990,

(3)HFD: a cholesterol-free high-fat diet with rodent food TD.06414 (Envigo, Barcelona, Spain) with the following percentages of calorie distribution: 21.4% carbohydrates, 18.3% proteins and 60 .3% fat (37% saturated, 47% monounsaturated and 16% polyunsaturated), and

(4)HFD-SB:HFD 250 mg/Kg of SB-204990.

At week 41 of life, mice were sacrificed after 16 hours of fasting for ex vivo analyses.

SB-204990 was purchased from Tocris and Sreeni Labs Private Limited.

metabolic tests

For the oral glucose tolerance test (OGTT), mice were fasted for 6 hours and received a dose of glucose (3 g/kg) by gavage.

For the intraperitoneal pyruvate tolerance test (IPPTT), mice were fasted for 6 hours and received an intraperitoneal injection of sodium pyruvate (2 g/kg).

For the insulin tolerance test (ITT), mice were fasted for 3 hours and received an intraperitoneal injection of insulin (1.5 IU/kg).

For the homeostatic model evaluation of insulin resistance index (HOMA-IR), the determinations were made after 16 hours of fasting. To determine glucose levels, blood samples were taken by venipuncture using a Precision Xceed glucometer (Abbott, Madrid, Spain). Insulin was measured in plasma using ELISA kits (Crystal Chem, Downers Grove, IL, USA).

RNA isolation and semiquantitative RT-PCR

Total RNA was isolated from frozen tissues using the easyblue total RNA extraction kit (number 17061, iNtRON Biotechnology, Inc., Seongnam, Korea). RNA concentration and quality were determined with the NanoDrop® ND-100 spectrophotometer. Total RNA (0.5-2 ^g) was used to synthesize cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories). mRNA expression was calculated using the 2-AACT method and normalized to Rps29 expression.

ACLY activity

ACLY activity was determined as described in MacDonaldet al.,2013. Liver tissue was lysed in ice-cold 220 mM mannitol, 70 mM sucrose, 5 mM potassium HEPES buffer, pH 7.5 containing 1 mM dithiothreitol. Lysates were centrifuged at 600xg for 10 minutes to precipitate nuclei and debris. The supernatant was then centrifuged at 5500 xg for 10 minutes to precipitate the mitochondrial fraction and then the supernatant was centrifuged at 20000 xg for 20 minutes to generate a cytosolic fraction used to measure enzyme activity. Enzyme activity was measured in 5 mM citrate, 0.3 mM coenzyme A, 3 mM ATP, 0.15 mM NADH, 10 mM MgCl2, 10 mM dithiothreitol and 6 units/ml malate dehydrogenase in 100 mM Tris chloride buffer, pH 8. 5 to 37°C. The disappearance of NADH was monitored at 340 nm for 1 min for background, after which 5 mM citrate was added to determine ACLY activity.

Primary hepatocyte isolation

Primary hepatocytes were isolated from the livers of wild-type male mice as described in López-Noriegaet al., 2019, with minor modifications. Livers were perfused with Hank's balanced salt solution (5.33 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 138 mM NaCl, 4.17 mM NaHCO3, and 25 mM HEPES) containing 5 mM glucose supplemented with 0.5 mM EGTA and 25 mM HEPES (pH 7.4 at 37°C) using a CTP100 peristaltic pump (ThermoFisher). Once the liver was perfused, the perfusion solution was changed to DMEM (Sigma-Aldrich D5546) supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (pen-strep), 10 mM HEPES, and 100 U/mL collagenase (type IV, Worthington). The cells were released from the liver and the cell suspension was filtered using a 70 ^M cell filter and centrifuged at 50 g x 2 minutes 3 times. Cell pellets were resuspended in DMEM (Sigma-Aldrich D5796) supplemented with penstrep, 10 mM HEPES, 10 nM dexamethasone, 2 mM l -glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS) and plated on plates previously coated with collagen. I (Sigma-Aldrich St. Louis, MO, USA). 60 min after plating, the medium was changed to DMEM (Sigma-Aldrich D5546) supplemented with pen-strep, 10 mM HEPES, 100 nM dexamethasone, and 10% FBS. The medium was changed 3 hours later to serum-free DMEM (Sigma-Aldrich D5546) supplemented with pen-strep, 5 mM HEPES, 10 nM dexamethasone, 2 mM l-glutamine, 1 mM sodium pyruvate and the cells were cultured for a maximum of 16 hours for several experiments.

Obtaining and culture of pancreatic islets

Pancreatic islets were isolated from wild-type mice by intraductal collagenase perfusion as described in Jimenez-Morenoet al.,2015. Islets were cultured for 16 hours in RPMI 1640 supplemented with 10% FBS, pen-strep, 2 mM glutamine, 1 mM sodium pyruvate, 50 ^M p-mercaptoethanol, and 10 mM HEPES.

Incorporation of glucose into lipids

Glucose incorporation into lipids was determined in primary hepatocytes, as described in Martin-Montalvoet al., 2016, with minor modifications. Primary hepatocytes were seeded in 12-well culture plates at 2.5 x 105 cells per well and incubated for 16 hours under the different experimental conditions. After a 60-minute preincubation in 20 mM HEPES, pH 7.4, 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 22.5 mM CaCl, 3 mM glucose, and albumin 1% fatty acid-free bovine serum at 37°C, cells were treated with 30,000 cpm [3-3H] glucose (0.5 Ci/mol, Perkin Elmer NET331C005MC) and plates were sealed with optical adhesive film . The incubation was terminated 2 hours later by adding 1 ml of methanol:phosphate-buffered saline (PBS) (2:3) to the cells. Cells were collected by gentle pipetting, centrifuged at 700 × g, and washed twice with PBS. 200 μl of 0.2 M NaCl was added to the cell pellet and the mixture was immediately frozen in liquid N2. The samples were thawed and a mixture consisting of 750 μl of CHCl3:methanol (2:1) and 50 μl of 0.1 N KOH per sample was added. Vigorous vortexing was then applied and the lipid and aqueous fractions were separated by centrifugation at 2000 × g for 20 minutes. The upper aqueous layer was discarded and the lower lipid-soluble layer was washed with 200 μL of methanol:water:CHCl3 (48:47:3). Aliquots (200 μl) of the lipid-soluble phase were transferred to scintillation vials and radiolabeled lipids were quantified using a liquid scintillation cocktail (Perkin Elmer).

cell death

Cell death was assessed by ELISA according to the manufacturer's instructions (Sigma-Aldrich 115446775001). Optical density at 405 nm versus the 490 nm reference was determined with a Variouskan Flash spectrophotometer (Thermo Scientific). In the case of primary hepatocytes, cells were seeded in 12-well plates. In the case of pancreatic islets, 10 islets per well were used.

Try MTT

The MTT test (colorimetric assay to measure the activity of cellular enzymes) was performed using the Cell Proliferation Kit I according to the manufacturer's recommendations (Roche, Spain). The optical density was determined at 575 nm with a reference wavelength of 690 nm using a Variouskan Flash spectrophotometer (Thermo Scientific, Spain). For primary hepatocytes, cells were seeded in 12- or 24-well plates. In the case of pancreatic islets, 35 islets per well were used.

urea test

Urea production was determined in primary hepatocytes, as described in Yeet al.,2017, with minor modifications. Primary hepatocytes from wild-type mice were seeded in 12-well plates at a density of 3 × 10<5>cells per well and incubated for 16 hours under the different experimental conditions. The medium was then replaced and urea production was measured after 2 hours using MAK006 kits according to the manufacturer's instructions (Sigma-Aldrich St. Louis, MO, USA) using a Variouskan Flash spectrophotometer (Thermo Scientific). .

Glucose-stimulated insulin secretion

Groups of 10 pancreatic islets were washed in 500 ^L Krebs-Ringer bicarbonate-HEPES (KRBH) buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH<2>PO<4>, MgSO<4> 0.5 mM, 21.5 mM CaCl, 32 mM NaHCO, 10 mM HEPES, 0.1% BSA and preincubated at 37°C for 45 minutes in 300 μl of KRBH. The islets were then centrifuged and the KRBH buffer was discarded. Subsequently, fresh KRBH supplemented with 2.2 mM glucose was added and the islets were incubated for 30 min. The buffer (basal insulin secretion) was then collected and 500 ^L of KRBH supplemented with 22 mM glucose was added. The islets were incubated for 30 minutes at 37°C and then the buffer was collected (stimulated insulin secretion). Insulin levels were measured using a mouse insulin ELISA kit according to the manufacturer's instructions (Crystal Chem).

Pharmacokinetic studies

Wild-type mice received oral gavage SB-204990 (30 mg/kg body weight) and serum samples were collected at different time points. SB-204990 in the serum samples was quantified by a chromatography-mass spectrometry method. 150 μl of acetonitrile containing 2,4,dichlorohydrocinnamic acid (internal standard) was added to 50 μl of serum samples, which were vortex mixed and centrifuged at 20,000 × g at 4°C for 15 minutes. 110 μl of supernatant was transferred to the autosampler vial for analysis.

Chromatographic experiments were carried out on an Agilent 1290 HPLC system (Agilent, CA). Separation of SB-204990 was performed using a Supelco 50 x 2.1 mm 3 ^m C18 column configured at 4°C. The mobile phase consisted of 90/10 water/acetonitrile with 0.1% formic acid as component A and 90/10 acetonitrile/water with 0.1% formic acid as component B using a linear gradient. Mass spectrometry analysis was performed using the Applied Biosystems/AB Sciex model API 4000 mass spectrometer system. Data were acquired and analyzed using Multiquant 2.1.1. (Sciex). Negative electrospray ionization data were acquired using multiple reaction monitoring (MRM). The standards were characterized using the MRM transitions (389/329) for SB-204990 and (218.9/174.8) 2,4, dichlorohydrocinnamic. The instrumental settings were 500°C and 4500 V and the composite parameter settings for the collision energy were -24eV for SB-204990 and -16eV for 2,4,didorohydrocinnamic.

Barnes Maze

The method was carried out in accordance with what was indicated in Patilet al., 2009. In the pretest, the mice were trained to enter the escape box by guiding them. The mice remained in the box for 2 minutes. Then, the training tests began (4 days). Mice were trained four trials per day and trials were separated by 15 minutes. Mice were allowed to explore the maze for up to 3 min and were guided to the escape box. Once the mice entered the box, the buzzer was turned off and the mice remained in the box for 1 min. The next day, subjects received a test trial for 90 seconds to determine short retention memory. During the test attempts the escape box was removed. Primary latency and total attempts were recorded. Without further training, 7 days later, the mice were tested in another test for 90 seconds to determine long-term memory retention.

Fecal lipid content

Lipid extraction from 1 g of dried feces was performed using chloroform-methanol as described in Hara and Radin, 1978.

Glycosylated hemoglobin

HbA1c levels were determined in blood samples according to the manufacturer's protocol (Crystal Chem).

Cholesterol and LDL/VLDL determinations

Serum cholesterol and low-density/very low-density lipoproteins (LDL/VLDL) were determined using the EnzyChrom assay kit (BioAssay Systems, E2HL-100) according to the manufacturer's instructions.

Rotarod

Results from rotarod tests are presented as the time it takes for mice to fall from an accelerating rotarod (4 to 40 rpm for 5 minutes). Mice were given a 1-min habituation test at 4 rpm the day before the experiment. The results shown are the averages of three trials per mouse (Soriaet al.,2019).

Cable suspension test

For the cable suspension test, mice were allowed to grasp a 1 mm horizontal cable with four legs for up to 60 seconds and the latency to fall from the cable was determined. Three different tests were carried out with each mouse and the results shown are the averages of the three tests (Soriaet al.,2019).

Histochemistry and cytochemistry

Dissected tissues and cell cultures were fixed in 4% paraformaldehyde. Tissue sections (5^m thick) were deparaffinized and rehydrated as described in López-Noriegaet al.,2019. For hematoxylin and eosin staining, sections were immersed in hematoxylin (Merck) and eosin (Merck) for 4 and 2 minutes, respectively. Evaluation of lipid content was performed by oil red O staining. Quantification of lipid content was performed after isopropanol solubilization at 510 nm. The histological study of the stained sections was performed with a Leica DM6000B microscope equipped with a DFC390 camera (Leica, Barcelona, Spain) and an Olimpus IX71 with an Olimpus DP70 camera (Olimpus). Adipocyte size was quantified with ImageJ software (NIH).

AML12 cell culture

Alpha 12 (AML12) mouse liver cells were obtained from the American Type Culture Collection (ATCC) and maintained and propagated in DMEM/F-12 Ham nutrient mixture with 10% fetal bovine serum, liquid media supplement. ITS (Sigma-Aldrich) and dexamethasone 0.1 ^M. For experiments, cells were exposed to 10% dialyzed FBS in DMEM/F-12 Ham nutrient mixture containing 17 mM glucose and treated with additional glucose (up to 42 mM), citrate, acetate, and SB-204990 for 16 hours. Samples were flash frozen and kept at −80°C until processing.

Extraction of acidic histones

AML12 cell pellets (2 x 106 cells) and liver samples were cut into small pieces (60-70 mg) and transferred to an Eppendorf tube. Subsequently, 1 ml of Triton Extraction Buffer (TEB; PBS containing 0.5% Triton of 50 mM sodium, with protease and phosphatase inhibitors P0044, P5725, P8340) per 200 mg of tissue/1 x 107 cells, and the samples were homogenized. The lysates were then incubated in rotation for 10 min at 4°C and the mixture was centrifuged at 2000 rpm for 10 min at 4°C. The supernatant was removed and the pellet was resuspended in 100 μl of 0.2 N HCl and incubated in rotation overnight at 4°C. Subsequently, the samples were centrifuged at 2000 rpm for 10 minutes at 4°C and the supernatant was transferred to a new tube. The HCl was then neutralized with 0.2 N NaOH and the samples were stored at −80°C until use.

western blot

For total protein isolation, samples were lysed in radioimmunoprecipitation assay buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate). pH 7.5) with protease, phosphatase and deacetylase inhibitors P0044, P5725, P8340 and SC-362323. Western blots were performed according to standard methods, which involved in certain cases membrane separation and incubation with a primary antibody of interest, followed by incubation with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. Blots were quantified with ImageJ, and bands of interest were normalized to Ponceau S, p-actin, and/or Gapdh staining, as previously validated in Jimenez-Gomezet al.,2013.

Determination of Ac-CoA

Ac-CoA was determined using the MAK039 kit following the manufacturer's instructions (Sigma-Aldrich).

Untargeted metabolomics

Metabolite extraction was performed on frozen tissues using 400 ^L MeOH:water (8/1, v:v). After vortexing, samples were subjected to five repeated freeze-thaw cycles and kept on ice for 1 h. The samples were then centrifuged (10 min, 15,200 rpm at 4°C) and 100 ^L and 250 ^L of supernatant were transferred to autosampler vials for respective liquid chromatography/mass spectrometry (LC/MS) and chromatography. gas/mass spectrometry (GC/MS).

For LC/MS and MS/MS, samples were injected into a UHPLC system (1290 Agilent) coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (6550 Agilent Technologies) operated in positive electrospray ionization (ESI+) mode. ) and negative electrospray (ESI-). Tissue extracts were separated using a Waters Acquity UPLC BEH HILIC (1.7 ^m, 2.1 x 150 mm). The solvent system was A = 50 mM ammonium acetate in water and B = acetonitrile/water with 50 mM ammonium acetate. The linear gradient elution used started with 95% A (time 0-2 min) and ended with 95% B (9.5 min). The injection volume was 2 ^L. The ESI conditions were 150°C gas temperature, 11 L/min drying gas, 35 psig nebulizer, 120 V fragmentator, and 65 V skimmer. The instrument was configured to acquire in the m/z 50 range. -1200 with an acquisition rate of 3 spectrum/s. MS/MS was performed in targeted mode, with a default isowidth (the half-maximum width of the quadrupole mass bandpass used during MS/MS precursor isolation) of 1.3 m/z (narrow). The collision energy was set at 10, 20, 30, and 40 V.

For GC/MS analysis, samples were dried under a stream of N2 gas and lyophilized before chemical derivatization with 40 ^L methoxyamine in pyridine (30 ^g/mL) for 45 minutes at 60°C. Subsequently, the samples were silylated using 25 ^L of N-methyl-Ntrimethylsilyltrifluoroacetamide with 1% trimethylcorosilane (Thermo Fisher Scientific) for 30 minutes at 60°C to increase the volatility of the metabolites. A 7890B GC system coupled to a 7250 QTOF mass spectrometer (Agilent Technologies) was used for GC-MS analysis. The derivatized samples (1 ^L) were injected into the gas chromatography system equipped with an Agilent 19091S-433UI HP5-ms Ultra Inert 86 stationary phase column (30 m x 0.25 mm x 0.25 ^m). Helium was used as carrier gas at a flow rate of 1.5 ml/minute in constant flow mode. The temperature gradient used was: from 70 to 190°C at a heating rate of 11°C/m, and from 190°C to 325°C at 21°C/min, finally holding for 4 minutes. Electron impact ionization was performed at 70 eV and the source temperature was set at 230°C. Mass spectra were recorded after a 3 min solvent delay, with the analyzer acquiring in full scan MS mode at a rate of 5 scans/s over a mass range of 35-700 m/z. At the time of acquisition, raw files from proprietary LC/MS vendors were converted to the open standard mzML format using Proteowizard MS-convert and subsequently processed with XCMS software (version 3.10.2). The specific retention period). Quality control samples were used to account for analytical variation.

In LC/MS experiments, features significantly modified by SB-204990 in STD and HFD were prioritized for MS/MS identification in specific mode. The metabolites were identified according to Level 2, as specified by Schymanskiet al.,2014 since their exact experimental and mass MS/MS spectra match the fragmentation pattern of the chemical standards of the METLIN, MassBank databases and/or NIST17. For GC/MS analyses, raw GC/MS data were converted to mzXML format using Proteowizard MS-convert. Subsequent data deconvolution and alignment were performed using eRah and metabolite identification was carried out by comparing the fragment spectra with the compound spectra present in the Golm database and NIST17 libraries. Data processing was performed in the R versions 3.6.1 and 4.0.3 (R-Foundation for Statistical Computing, www.Rproject.org).

Liver lipid analysis

Hepatic lipids were extracted from ~20 mg of tissue using hexane-isopropanol as described in Hara and Radin, 1978. Samples were analyzed by GLC as described in Martinez-Forceet al., 2004. Fatty acid methyl esters (FAMES) were obtained from isolated lipids by heating the samples at 80°C for 1 hour in 3 ml of methanol/toluene/H2SO4 (88:10:2 v/v). Heptadecanoic acid (1/10 w/w) was added to each sample as an internal standard to allow quantification. After cooling, 1 ml of heptane was added and the samples were mixed. FAMES were recovered from the upper phase and then separated and quantified using a Hewlett-Packard 5890A gas chromatograph (Palo Alto, CA, USA) with a Supelco SP-2380 fused silica capillary column (30 m length , 0.25 mm film thickness) (Bellefonte, PA, USA). Hydrogen was used as the carrier gas, with a linear gas rate of 28 cm/s. The detector and injector temperatures were set at 220°C and the oven at 170°C, with a split ratio of 1:50. Fatty acids were identified using standards (Sigma-Aldrich, St. Louis, MO, USA). Triacylglycerides and cholesterol esters were separated and quantified by GLC as described in Fernandez-Moyaet al.,2000 with an Agilent 6890 gas chromatograph (Palo Alto, CA, USA). Triheptadecanoic acid was added to the samples as an internal standard for quantification. The temperatures of both the injector and the detector were 380°C, the oven temperature was 345°C and a head pressure gradient of 70 to 120 kPa was applied, changing this last parameter depending on the column. The gas chromatography capillary column was a J & W Scientific DB-17HT (15 m length, 0.25 mm i.d., 0.15 ^m film thickness) (Folsom, CA, USA), with a linear gas rate of 50 cm/s, the split ratio was 1:80, and the detector was a flame ionization detector (FID). The different triacylglycerides and cholesterol esters were identified with respect to known samples, and the FID response was corrected. Polar lipids were analyzed and quantified by HPLC. HPLC separation was carried out on a Waters 2695 Module (Milford, MA) equipped with a Waters 2420 ELSD. The column used was a Lichrospher 100 Diol 254-4 (5 ^m; Merck) applying a method based on a binary linear gradient of solvent mixtures containing different proportions of hexane, 2-propanol, acetic acid, water and trimethylamine. The flow rate was 1 ml/min, the data were processed with Empower software, and the ELSD was regularly calibrated with high-purity commercial standards for each lipid.

Oxygen consumption and ECAR

To study mitochondrial bioenergetics in primary hepatocytes, cells were measured using an XF24 extracellular flux analyzer (Agilent). After 16 hours of treatment, the cells were washed with Seahorse assay medium (Seahorse Bioscience), which was supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH 7.2. The cells were incubated in an incubator without CO2 at 37°C for 1 hour. Then, the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured. OCR and ECAR were determined under basal conditions and by consecutive injections of oligomycin (4 ^M) at minute 27, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 2 ^M) at minute 52, rotenone (1 ^M ) at minute 78 and antimycin A (5 ^M) at minute 104. OCR rates were determined in fresh liver mitochondria in samples from mice exposed to an oral gavage of SB-204990 (30 mg/kg body weight ) following the Agilent isolated mitochondria protocol. Briefly, 3 hours after gavage, the liver was removed and the square lobe was transferred to a GentleMACS™ dissociation tube with 5 ml of ice-cold MSHE buffer (70 mM sucrose, 210 mM mannitol, 5.0 mM HEPES , 1.0 mM EGTA, 0.5% (w/v) fatty acid-free BSA, pH 7.2). Subsequently, the tissue was homogenized in a GentleMACS tissue dissociator under the mouse-mito tissue isolation cycling protocol. Fresh samples were filtered twice through a 40 ^m mesh filter and finally through a 10 ^m mesh filter. The filtrate was centrifuged at 9000g for 10 minutes at 4°C and the pellet was resuspended in 200 μl of ice-cold BSA-free MSHE to quantify protein content. Then, 0.5% (w/v) fatty acid-free BSA was added and the samples were diluted in warm (37°C) MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, HEPES 2 mM, 1 mM EGTA, 10 mM succinate, 2 ^M rotenone, 0.5% (w/v) fatty acid-free BSA, pH 7.2). 5 ^g of mitochondria were seeded per well on Seahorse plates and the plates were centrifuged at 2000g for 20 minutes at 4°C using a swing-bucket microplate adapter. OCR was determined by consecutive injections of ADP (4 mM) at minute 13, oligomycin (3.16 ^M) at minute 20, FCCP (4 ^M) at minute 26, and antimycin A (4 ^M) at the 31st minute.

Statistic analysis

Statistical analysis was performed using SigmaPlot 12.0 (SigmaPlot, Barcelona, Spain) and GraphPad prism 7 (GraphPad Software Inc, San Diego, CA). Different statistical tests were used based on the number of groups and recommendations of the statisticians SigmaPlot 12.0 and GraphPad prism 7. Statistical tests include one-way ANOVA, two-way ANOVA and student's t-test. In all cases, at least 3 biological replicates were included in the experimentation. Data are shown as means ± SEM. Significance is reported at p < 0.05.

Results

SB-204990 extends life expectancy and health

To define the age-dependent metabolic changes that contribute to promoting aging processes, as well as causing premature death, we evaluated different parameters of glucose homeostasis in healthy mice of different ages fed a standard diet (STD). Body weight and weight of various tissues was higher in adult and old mice (Figure 1A-B). Assessment of oral glucose tolerance indicated the absence of significant age-dependent alterations in glucose or insulin levels during tests carried out in young and old mice. However, significant differences were observed specifically in adult versus old mice in circulating insulin levels (Figure 1C-F). Analysis of pyruvate tolerance and insulin sensitivity indicated a reduced ability to promote hepatic gluconeogenesis, as well as severe insulin resistance in aged mice (Figure 1G-J). Under fasting conditions, old mice exhibited hyperinsulinemia, while maintaining normoglycemia, producing a higher homeostatic model assessment index of insulin resistance (Homa-IR) (Figure 1K-M). These data indicated that the most prominent effects of aging involve a deterioration in functionality in insulin target tissues in the control of glucose metabolism.

We next focused on determining whether ACLY expression levels could contribute to the aforementioned changes in glucose metabolism in metabolic tissues in an age-dependent manner.

Old mice exhibited higher levels of ACLY expression and enzyme activity in the liver (Figure 1N and Figure 2A). These data support the important role of hepatic ACLY in aging and corroborate that restricting ACLY expression and/or activity could restrain intrinsic aging processes.

SB-204990 improves metabolic and physical function in mice fed a high-fat diet (HFD)

Under physiological conditions, ACLY has been proposed as the main producer of cytosolic Ac-CoA (Pietrocolaet al., 2015). Cytosolic Ac-CoA is used for the production of endogenous lipids and for the production of malonyl-coenzyme A, an inhibitor of carnitine palmitoyltransferase I required for fatty acid uptake into the mitochondria (Schlaepfer and Joshi, 2020).

Incorporation of radiolabeled [H3]-glucose into lipids was measured to indirectly assess liponeogenesis in primary hepatocytes isolated from wild-type male mice. The results indicated that SB-204990 causes a dose-dependent inhibition of glucose-dependent de novo lipogenesis (Figure 4A). Cell death measured via ELISA and urea secretion indicated that toxicity occurs at concentrations greater than 10^M, with marked toxicity at 100^M (Figure 4B-C). Experiments on primary pancreatic islets isolated from wild-type mice indicated reduced susceptibility to SB-204990-promoted alterations in functional and viability tests compared to hepatocytes (Figure 4D-F).

Taken together, these results show that in vitro inhibition of fatty acid biosynthesis mediated by SB-204990 inhibition of ACLY can be achieved at non-toxic concentrations (i.e., ~10^M).

Pharmacokinetic studies were performed to determine circulating levels of SB-204990 following oral administration in mice. The results indicated that a single dose of 30 mg/kg body weight of SB-204990 generated a plasma concentration of ~ 4 ^M 2 hours after ingestion, in the range of non-toxic concentrations used in in vitro studies and at doses that prolonged lifespan in yeast and flies (Figure 3A).

These data were used to estimate the optimal dose of SB-204990 for long-term in vivo studies at 0.25 mg of compound/g of food (~ 4 g of food intake/day) in mice.

Wild-type mice were fed a healthy diet (STD) or a cholesterol-free diabetogenic high-fat diet (HFD) and treated or not with SB-204990 for 15 weeks. As expected, in vivo treatment with the ACLY inhibitor SB-204990 did not alter ACLY enzymatic activity in liver tissue (Figure 4G) (Pearceet al., 1998) and the body weight of STD-fed mice was lower than that of HFD-fed mice (Figure 3B).

HFD-fed and SB-204990-treated mice exhibited lower body weight beginning at week 9 of treatment compared to HFD-fed mice. Daily energy intake and fecal lipid content were not altered by SB-204990 supplementation (Figure 4H-I). However, fasting-induced energy intake was higher in mice on STD diet and treated with SB-204990 compared to their untreated peers (Figure 4J).

The effects of SB-204990 on glucose homeostasis were subsequently investigated. The oral glucose tolerance test (OGTT) indicated that, although animals fed the same diets started with similar values, SB-204990 produced a mild impairment in glucose tolerance in STD-SB mice, while in Mice fed HFD-SB had the opposite effect, that is, an improvement, compared to untreated HFD (Figure 3C-D). Similar results were obtained in an intraperitoneal pyruvate tolerance test (IPPTT), indicating a critical role for ACLY in liver tissue (Figure 3E-F).

To evaluate whether changes in glucose tolerance are due to inefficient functioning of pancreatic p cells or resistance to insulin action in their target tissues, an insulin tolerance test (ITT) was performed (Figure 3G-H).When given an intraperitoneal insulin injection, all groups showed a deficit in their responses compared to the STD group. Determination of insulin levels during an oral glucose tolerance test (OGTT) indicated that STD-SB responds with potent insulin secretion, suggesting that insulin resistance is the main underlying cause of insulin homeostasis. dysregulated glucose in STD-SB mice (Figure 3I-J). HFD-SB mice exhibited similar insulin levels during OGTT. Evaluation of glucose levels during a 24-hour fasting period indicated that STD-SB mice exhibited higher glucose levels compared to STD mice, while HFD-fed mice exhibited similar levels regardless of supplementation. SB-204990 (Figure 3K-L). Glucose levels and insulin levels under fasting conditions were higher in STD-SB mice compared to STD-fed mice, resulting in a higher HOMA-IR index (Figure 5A-C). In contrast, HFD-fed mice treated with SB-204990 exhibited lower levels of fasting glucose, fasting insulin, and lower HOMA-IR values compared to their HFD-fed counterparts (Figure 5A-C). The percentage of glycosylated hemoglobin indicated a trend toward reduced levels in mice fed HFD-SB compared to mice fed HFD (p = 0.07 in two-way ANOVA), with no differences in mice fed a healthy diet (Figure 5D ).

To elucidate whether SB-204990-mediated ACLY inhibition influences cholesterol homeostasis, circulating low-density/very-low-density lipoprotein (LDL/VLDL) levels and total cholesterol levels were measured (Figure 5E-F). . Administration of SB-204990 under HFD reduced circulating levels of LDL/VLDL and total cholesterol, while no changes were detected in SB-204990-treated mice under STD compared to their untreated counterparts. Surprisingly, in both cases, the values in the HFD-SB group were comparable to those in healthy fed STD mice, demonstrating that SB-204990 prevents the deleterious effects of HFD on cholesterol homeostasis.

Next, we focused on determining the effects on motor function outcomes. SB-204990 in HFD improved physical performance in the Rotarod performance tests (which measures coordination, balance, and fatigue in rodents) and Wire Hang (which evaluates motor and central nervous system (CNS)) function. in rodents), indicating the healthier overall condition of these animals compared to HFD-fed mice (Figure 5G-H). Spatial memory was not altered by SB-204990 supplementation regardless of diet (Figure 6A-F).

At the time of sacrifice (i.e., 15 weeks of treatment), STD-fed mice treated with SB-204990 exhibited greater white adipose tissue (WAT) mass, while Mice fed HFD supplemented with SB-204990 showed reduced WAT mass compared to their respective untreated diet (Figure 5I). Histological examination of the tissues indicated a massive reduction in lipid content in liver tissue and smaller adipocyte size in mice treated with HFD-SB, while no significant effects were observed in skeletal and pancreatic muscle sections (Figure 5J-K). Similar to mice lacking ACLY in white adipose tissue (Zhaoet al., 2016), healthy fed mice treated with SB-204990 exhibited increased mean adipocyte size in white adipose tissue.

Taken together, these results show that SB-204990 has opposite effects depending on the diet provided. In healthy STD-fed mice, SB-204990 results in impaired glucose homeostasis due to insulin resistance, suggesting that a certain level of ACLY activity is required to maintain metabolic health. However, under metabolically challenging conditions, such as an HFD diet, SB-204990-promoted ACLY inhibition improves metabolic and physical health.

SB-204990-induced metabolic reprogramming is independent of global changes in histone acetylation

The contribution of ACLY in cellular reprogramming under different metabolic conditions was evaluated, specifically whether the restricted activity of ACLY promoted by SB-204990 could alter histone acetylation, a global mechanism that controls transcription (Wellenet al., 2009). Incubation of AML12 hepatocyte cells with acetate, a substrate of cytoplasmic Ac-CoA synthetase (Acs, also known as AceCS1) to produce cytosolic Ac-CoA, in the order of concentrations in the portal vein (Balmeret al., 2016 ; Zhaoet al.,2020), produced maximum levels of histone acetylation in all core histones tested (Figure 7A and Figure 8A). Surprisingly, equimolar abundance of the ACLY substrate citrate did not enhance histone acetylation, suggesting that citrate availability does not limit acetylation events.

Supraphysiological glucose levels, which could promote Ac-CoA generation through the mitochondrial metabolism of glycolysis, did not result in consistent increases in histone acetylation, demonstrating that net increases in histone acetylation could depend more of the cytoplasmic activity of Acs than of the activity of ACLY. Under these cellular conditions, SB-204990 treatment did not decrease histone acetylation levels (Figure 7A and Figure 8A). The differences between the effects of acetate, citrate and glucose, and the lack of effects of SB-204990, support the idea that cytoplasmic Acs is an important source of Ac-CoA in promoting histone acetylation events that regulate chromatin structure in AML12 hepatocytes. In mouse liver, analysis of histone acetylation, global protein acetylation levels, and total Ac-CoA levels confirmed the lack of differences in SB-204990-treated mice fed a healthy diet or HFD ( Figure 7B-F). These data suggest that compensatory mechanisms enhance Ac-CoA generation following SB-204990-mediated ACLY inhibition. Therefore, modulation of global protein acetylation (including histones) is not the primary cause of the metabolic effects of SB-204990.

Cholesterogenesis is among the main effectors of the action of SB-204990

Analysis of hepatic cholesterol esters indicated a robust reduction in both SB-treated STD-fed and SB-treated HFD-fed mice (Figure 9A). Global analysis of hepatic fatty acids indicated minor differences in fatty acid species, revealing a slight reduction in palmitic acid in STD-SB mice and a modest increase in vaccenic acid in HFD-SB mice (Figure 9D). Accordingly, analysis of triglyceride species revealed a specific decrease in triglycerides containing two palmitic acids (POP and PLP) in STD-SB mice (Figure 9E). In HFD-SB mice we found higher hepatic levels of several species of triglycerides containing two saturated fatty acids (POP, PLS) and lower levels of triglycerides containing polyunsaturated fatty acids (POL, PLL OOL), an effect that has been observed in ACLY-depleted LNCaP cells (Figure 9E)(Migitaet al.,2014). Analysis of polar lipid species indicated reduced global phosphatidylserine levels in STD-SB mice. Analysis of modulated hydrophobic species in our untargeted LC/MS metabolomics indicated increased levels of several long-chain phosphatidylcholine species, while short-chain phosphatidylcholine species were decreased (Figure 9F). Furthermore, alterations in diglyceride and phosphatidylinositol species were also detected, indicating profound modulation in the lipidome (Figure 9F). Surprisingly, general agreement with these trends was also found in HFD-fed SB-204990-treated mice (Figure 9F). Furthermore, we observed a robust reduction of pteridine and betaine precursors related to the folate cycle and metabolism of a carbon and a concomitant increase in transmethylated carnitine in STD-fed mice treated with SB-204990 (Figure 9G). Of note, glycerol 3-phosphate, a precursor in glycerolipid synthesis, was increased in the liver of STD-SB mice (Figure 9G). Interestingly, SB-204990 failed to affect these metabolite levels in mice fed an HFD. These data suggest that SB-204990-induced modulations in the folate cycle are more prominent under healthy feeding conditions, revealing differences in how this pathway is activated under different feeding regimes.

Overall, the results shown revealed that SB-204990 produces modulations in pathways related to improved survival and health, including the metabolism of various amino acids and lipids (Annibalet al.,2021; Aonet al.,2020).

SB-204990 targets liver mitochondrial function

Contrary to the putative role of ACLY in the production of Ac-CoA for de novoy fatty acid synthesis despite the fact that glucose incorporation into lipids was reduced by SB-204990, we found that primary hepatocytes exhibited a rapid increase in the lipid content after treatment with SB-204990 (Figure 10A-B and Figure 4A). Interestingly, similar effects (e.g., larger lipid droplets/adipocyte size) have been observed in healthy fed mice and cell cultures without ACLY activity (Figure 5J-K) (Martinez Calejmanet al., 2020). Incubation of primary hepatocytes with acetate or citrate did not increase lipid content (Figure 10C and Figure 11A). Culture conditions mimicking hyperglycemia showed a trend toward greater lipid accumulation that was statistically significant only when supplemented with acetate. . Under basal glucose conditions, SB-204990 produced net increases in lipid content even when culture media were supplemented with citrate and acetate. Lipid accumulation under conditions of SB-204990-mediated ACLY inhibition could be explained by the restricted metabolic activity. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test indicated that, under basal conditions, SB-204990 reduces cellular metabolism (Figure 10D). Surprisingly, acetate supplementation did not restore the metabolic activity of primary hepatocytes treated with SB-204990, supporting previous observations indicating that the restricted activity of ACLY cannot be fully compensated by acetate supplementation (Zhaoet al., 2016).

The effect of SB-204990 on mitochondrial performance, which could enhance lipid accumulation, was studied. To this end, mitochondrial functional dynamics was evaluated in primary hepatocytes treated with SB-204990. SB-204990 reduced the basal and maximal oxygen consumption rates (OCR), while specifically the maximal consumption rates were decreased in high glucose (25 mM) cultured cells treated with SB-204990 (Figure 10E). In standard and high glucose content media, SB-204990 promoted the reduction of extracellular acidification rate (ECAR) in cells challenged with oligomycin and FCCP, indicating reduced glycolysis (Figure 10F). This result suggests allosteric inhibition of glycolysis by intracellular accumulation of citrate (Garlandet al., 1963). Next, we determined whether SB-204990 could elicit effects on mitochondria in vivo. Freshly isolated liver mitochondria from mice treated with SB-204900 (30 mg/kg body weight; oral gavage; 3 hours of treatment) exhibited a block and a restricted increase in OCR induced by ADP and FCCP, respectively (Figure 10G). These data indicate a rapid effect of SB-240990 on mitochondrial uncoupling and suggest that factors involved in energy storage and utilization could be modulated under conditions of SB-204990-mediated ACLY inhibition. Interestingly, we found that pThr 172 Ampk phosphorylation was reduced in the liver of mice treated for 15 weeks with SB-204990 regardless of diet, as well as in primary hepatocytes treated with SB-204990, suggesting an energy-rich state ( Figure 10H and Figure 11B-C). Primary hepatocytes were simultaneously treated with SB-204990 and the Ampk activator metformin (Figure 10I-K). Metformin normalized metabolic activity and lipid levels, indicating that these effects require blockade of Ampk activity. Similar results were obtained using the Ampk activator AICAR (Figure 11D). Importantly, pharmacological inhibition and activation of other pathways altered by SB-204990, such as mTOR and the folate cycle, did not reverse the alterations in MTT activity and lipid content promoted by SB-204990, indicating an important role of Ampk inhibition in these effects (Figure 11D). In liver tissue, pThr 172 Ampk restricted was associated with restricted mTOR signaling markers and increased Sirtl expression in STD-SB-204990 mice, while that mTOR signaling was found to be unaltered and increased Nmnat expression occurred in HFD-SB-204990 mice (Figure 10H and Figure 11C). These results suggest that enhanced Sirt1 expression or activity, through increased NAD+ availability and/or restricted mTOR activity, could compensate for reduced Ampk activity (Aonet al.,2020; Gomeset al.,2013). Importantly, markers of mitochondrial content and biogenesis were reduced in the liver of SB-204990-treated mice fed a healthy diet, while expression levels of Acaa2, involved in mitochondrial p-oxidation, increased in both mice. fed with both STD and HFD treated with SB-204990 (Figure 10H and Figure 11C).

Modulations in mitochondrial functional dynamics could alter stress resistance and the accumulation of oxidative damage (Martin-Montalvoet al.,2013). Liver lysates from mice treated with SB-204990 exhibited reduced levels of response markers to DNA damage (STD-SB) or lipid peroxidation (HFD-SB), as well as increased expression of the antioxidant protein Sod1, while Autophagy markers exhibited divergent effects (Figure 11E-F). Under healthy feeding conditions, Fas levels were reduced, supporting the idea that reduced mitochondrial metabolism might have a greater specific weight promoting lipid accumulation in vivo than alterations in de novo lipogenesis (Figure 10H and Figure 11C). . All these results indicate that ACLY is at the crossroads of regulating molecular mechanisms of aging, such as liponeogenesis and mitochondrial metabolic centers, and plays an important role in the control of energy metabolism through Ampk signaling.

In view of the results presented here, it is demonstrated that treatment with the ACLY inhibitor SB-204990 extends life in several experimental models and improves metabolic and physical health in diet-induced obese mice, which generally suffer premature death.

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Claims (16)

1. ATP-citrate lyase inhibitor for use as a geroprotective agent in a patient population that presents altered, pathological, or levels outside those considered healthy, of one or more of the metabolic parameters selected from the list that consists of: glucose level, pyruvate level, insulin level, low and very low density lipoprotein level, cholesterol level and lipid content in liver tissue. 2. The ATP-citrate lyase inhibitor according to the previous claim where the inhibitor is SB-204990, BMS 303141, bempedoic acid, NDI-091143, hydroxycitric acid, 2-furoic acid, Litarex, MEDICA16, or any of their combinations. 3. The ATP-citrate lyase inhibitor according to any of the preceding claims for use in the treatment of metabolic diseases. 4. The ATP-citrate lyase inhibitor according to the previous claim wherein the metabolic disease affects tolerance to glucose, pyruvate and/or insulin. 5. The ATP-citrate lyase inhibitor according to any of claims 3 - 4 wherein the metabolic disease affects the blood levels of low and very low density lipoproteins and/or cholesterol. 6. The ATP-citrate lyase inhibitor according to any of claims 3 - 5 wherein the metabolic disease affects the lipid content in the liver tissue. 7. The ATP-citrate lyase inhibitor according to any of claims 3 - 6 wherein the metabolic disease occurs with an increase in body weight, an increase in the mass of white adipose tissue and/or an increase in the size of adipocytes. 8. The ATP-citrate lyase inhibitor according to any of claims 3 - 7 wherein the metabolic disease is selected from the list consisting of diabetes and fatty liver. 9. The ATP-citrate lyase inhibitor according to any of the preceding claims for use in improving functional capacity. 10. The ATP-citrate lyase inhibitor according to the previous claim where the improvement in functional capacity refers to the improvement in the results of tests that measure the strength, resistance and/or motor coordination of the individuals. 11. The ATP-citrate lyase inhibitor according to any of the preceding claims for use in improving neurocognitive health. 12. The ATP-citrate lyase inhibitor according to the previous claim wherein the improvement in neurocognitive health refers to the improvement in the results of tests that measure the memory of individuals. 13. A composition comprising ATP citrate lyase inhibitors according to any of claims 1-12 for use as a geroprotective agent. 14. The composition according to the preceding claim further comprising an Ampk activator. 15. The composition according to the previous claim wherein the Ampk activator is metformin and/or AICAR. 16. The composition according to any of claims 13-15, which is a pharmaceutical composition.