British Journal of Pharmacology 2015 Oct;172(20):4996-5008. doi: 10.1111/bph.13269.

Combining citrulline to atorvastatin preserves glucose homeostasis in a murine model of diet-induced obesity.

 

Frédéric Capel1, Gwladys Chabrier1, Elodie Pitois1, Jean-Paul Rigaudière1, Servane Le Plenier2, Christine Durand4, Chrystèle Jouve1, Jean-Pascal de Bandt2,3, Luc Cynober2,3, Christophe Moinard2, Béatrice Morio1,4

1 INRA, UMR 1019, UNH, CRNH Auvergne and Clermont University, Auvergne University, Human Nutrition Unit, Clermont-Ferrand, France

2 Laboratory of Nutrition Biology EA 4466, Paris-Descartes University, Paris, France

3 Clinical Chemistry, Cochin and Hotel-Dieu Hospitals, Assistance Publique – Hôpitaux de Paris, Paris, France

4 INRA, UMR 1397, CarMeN Laboratory, Lyon 1 University, INSERM U1060, INSA of Lyon, Rockefeller and Charles Merieux Lyon-Sud Medical Universities, Lyon, France

 

Abstract

Statins are part of the world’s most-prescribed class of medications. They are the cornerstone treatment for dyslipidemia and prevention of cardiovascular diseases. They lower blood cholesterol by inhibiting the enzyme HMG-CoA reductase, which plays a central role in hepatic cholesterol production. We have demonstrated in a preclinical study that citrulline, a non-protein amino acid found in cucurbits and mainly synthesized by the intestine in animals, synergistically act with the statin, atorvastatin, to improve glucose homeostasis in a murine model of diet-induced obesity. Intra-abdominal fat mass was also decreased at the whole body level. Thus, citrulline is a relevant synergistic molecule to potentiate beneficial health effects of statins and limit part of its secondary effects. These findings thus open interesting perspectives based on pharmaco-nutritional strategies for prevention and management of obesity and type 2 diabetes.

 

 

Supplement:

Statins are used for the treatment of dyslipidemia and for the prevention of cardiovascular diseases. Statins have been shown to reduce the five-year occurrence of major cardiovascular events through reduction in plasma LDL-cholesterol (1). However, long-term treatment with statins is associated with alteration in glucose homeostasis and increased risk of developing type 2 diabetes (T2DM)(2). The risks for developing T2DM with statins are higher in individuals who meet criteria of metabolic syndrome compared to those who do not (3). Furthermore, the risk of new onset T2DM is increased with high-intensity statin treatment (4). Based on the METSIM cohort, a recent prospective study concluded the increased risk of T2DM associated with statin treatment may be due to a decrease in both insulin sensitivity and insulin secretion (5). In pancreatic beta-cells, statins appear to increase basal secretion and to alter glucose-induced secretion, which results in constitutive secretion of insulin (6-7). As the ability of statins to decrease major cardiovascular events and mortality outweighs the risk of incident T2DM, no change in clinical practice is currently recommended (8). However, it is clearly necessary to develop strategies to minimize the negative metabolic effects of statins and maintain glucose homeostasis, especially in pre-diabetic patients and in patients at high cardiometabolic risks.

In an in vitro assay with endothelial cells, co-treatment with atorvastatin and citrulline was shown to potentiate the production of nitric oxide (NO) (9). This effect was mediated through increased bioavailability of arginine, a metabolite of citrulline, as well as to enhanced activity of the NO generating enzyme eNOS. In addition to their ability to decrease cholesterol synthesis, statins increase eNOS expression and activity. As NO is a central regulator of energy and lipid metabolism, body composition and insulin sensitivity (10), and as NO homeostasis is compromised in obesity and T2DM (11), the co-treatment of atorvastatin and citrulline was tested in a murine model of diet-induced obesity and insulin resistance.

Male C57BL/6J mice (n=160) received a standard diet (STD) or a diet high in fat and sugar (HFHS, high-fat high-sugar) during 12 weeks. During the last three weeks of intervention, HFHS mice were separated into 4 groups to receive only HFHS diet, or HFHS diet enriched with citrulline (2.5 g/kg, HFHScit) or atorvastatin (10 mg/kg, HFHSstat) or the combination of the two (HFHScit-stat). Body composition, glucose tolerance, insulin sensitivity and hepatic lipid metabolism were examined at the end of experiment. The effect of the treatment was tested using one-way analyses of variance (ANOVA, Statview software, SAS Institute, USA) followed by post hoc Fisher LSD multiple comparison if significant. Differences were considered significant at p<0.05.

The HFHScit-stat group was the only one that demonstrate an improved body weight (-4%), fat mass (-29%), glucose tolerance (+ 85%) and insulin sensitivity (30%) in comparison to the other HFHS groups (p<0.05). In addition, postprandial insulin in mice fed HFHScit-stat was similar to STD animals and was 42% lower than that of the other HFHS groups (p<0.05), whereas postprandial glucose levels were not significantly different between groups. In the liver of HFHScit-stat mice, the level of AMPK phosphorylation was similar to STD-fed animals and was 27% higher than that of HFHS-fed animals (p<0.05). This was associated with a decreased lipogenic activity in response to meals in HFHScit-stat vs. HFHS groups (-81%, p<0.05). In addition, gene expression Srebf1, Lxralpha, Chrebp and target genes, Acc1, Fasn and Scd1, was significantly lower in fasted and fed HFHScit-stat mice compared to other HFHS groups (p<0.05).

We thus conclude that the combination of citrulline to atorvastatin synergistically act to reduce fat storage and restore glucose homeostasis in mice receiving a HFHS diet. In addition, this pharmaco-nutritional combination potentiates the activation of AMPK in the liver, thereby limiting the lipogenic activity.

The importance of this study is three-fold:

  • First, these findings demonstrate that combining citrulline to atorvastin can be a promising strategy to minimize the negative metabolic effects of the statin and maintain glucose homeostasis, especially in pre-diabetic patients and in patients at high cardiometabolic risks. This preclinical study must be however confirmed in a proof-of-concept clinical trial.
  • Second, our original scientific approach opens perspectives for the development of new pharmaco-nutritional strategies based on synergistic mechanisms between drugs and nutrients for the prevention and management of metabolic diseases, such as obesity and T2DM.
  • Third, our study points out for a need for enhancing our understanding of citrulline metabolism at the whole body and tissue-specific levels in obesity and T2DM. This area of research is essential to support the use of this amino acid in pathophysiological situations and to master our nutritional recommendations.

 

References

1- Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, et al.; Cholesterol Treatment Trialists’ (CTT) Collaborators. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet. 2005; 366:1267-78.

2- Food and Drug Administration (2012). FDA drug safety communication: important safety label changes to cholesterol-lowering statin drugs. Available from: http://www.fda.gov/Drugs/DrugSafety/ucm293101.htm. Accessed September 5, 2012.

3- Waters DD, Ho JE, Boekholdt SM, DeMicco DA, Kastelein JJ, Messig M et al. Cardiovascular event reduction versus new-onset diabetes during atorvastatin therapy: effect of baseline risk factors for diabetes. J Am Coll Cardiol. 2013;61:148-52.

4- Kohli P, Waters DD, Nemr R, Arsenault BJ, Messig M, DeMicco DA et al. Risk of new-onset diabetes and cardiovascular risk reduction from high-dose statin therapy in pre-diabetics and non-pre-diabetics: an analysis from TNT and IDEAL. J Am Coll Cardiol. 2015;65:402-4.

5- Cederberg H, Stančáková A, Yaluri N, Modi S, Kuusisto J, Laakso M. Increased risk of diabetes with statin treatment is associated with impaired insulin sensitivity and insulin secretion: a 6 year follow-up study of the METSIM cohort. Diabetologia. 2015 Mar 10.

6- Tsuchiya M, Hosaka M, Moriguchi T, Zhang S, Suda M, Yokota-Hashimoto H et al. Cholesterol biosynthesis pathway intermediates and inhibitors regulate glucose-stimulated insulin secretion and secretory granule formation in pancreatic beta-cells. Endocrinology. 2010;151:4705-16.

7- Ishikawa M, Okajima F, Inoue N, Motomura K, Kato T, Takahashi A et al. Distinct effects of pravastatin, atorvastatin, and simvastatin on insulin secretion from a beta-cell line, MIN6 cells. J Atheroscler Thromb. 2006;13:329-35.

8- Stone NJ, Robinson JG, Lichtenstein AH, Bairey Merz CN, Blum CB, Eckel RH et al.; American College of Cardiology/American Heart Association Task Force on Practice Guidelines. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014; 63:2889-934.

9- Berthe MC, Bernard M, Rasmusen C, Darquy S, Cynober L, Couderc R. Arginine or citrulline associated with a statin stimulates nitric oxide production in bovine aortic endothelial cells. Eur J Pharmacol. 2011; 670:566-70.

10- Sansbury BE, Hill BG. Regulation of obesity and insulin resistance by nitric oxide. Free Radic Biol Med. 2014; 73C:383-399.

11- Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Chayama K, Oshima T. Effect of obesity on endothelium-dependent, nitric oxide-mediated vasodilation in normotensive individuals and patients with essential hypertension. Am J Hypertens. 2001; 14:1038-45.

 

Acknowledgements:  The work was supported by the authors’ institutions, INRA and Paris-Descartes University.

 

Duality of interest: Christophe Moinard, Luc Cynober, Jean-Pascal de Bandt and Servane Le Plenier are shareholders of CITRAGE© (Créteil, France).

 

Contact:
Béatrice Morio, PhD
Research Director INRA,
CarMeN Laboratory
Faculté de Médecine LYON SUD – BP 12
165 Chemin du Grand Revoyet
69921 OULLINS CEDEX
France
beatrice.morio@lyon.inra.fr
http://carmen.univ-lyon1.fr

 

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