Effects of lipid-lowering drugs on irisin in human subjects in vivo and in human skeletal muscle cells ex vivo

Abstract

Context and objective: The myokine irisin has been proposed to regulate energy homeostasis. Little is known about its association with metabolic parameters and especially with parameters influencing pathways of lipid metabolism. In the context of a clinical trial, an exploratory post hoc analysis has been performed in healthy subjects to determine whether simvastatin and/or ezetimibe influence serum irisin levels. The direct effects of simvastatin on irisin were also examined in primary human skeletal muscle cells (HSKMCs).

Design and participants: A randomized, parallel 3-group study was performed in 72 men with mild hypercholesterolemia and without apparent cardiovascular disease. Each group of 24 subjects received a 14-day treatment with either simvastatin 40 mg, ezetimibe 10 mg, or their combination.

Results: Baseline irisin concentrations were not significantly correlated with age, BMI, estimated GFR, thyroid parameters, glucose, insulin, lipoproteins, non-cholesterol sterols, adipokines, inflammation markers and various molecular markers of cholesterol metabolism. Circulating irisin increased significantly in simvastatin-treated but not in ezetimibe-treated subjects. The changes were independent of changes in LDL-cholesterol and were not correlated with changes in creatine kinase levels. In HSKMCs, simvastatin significantly increased irisin secretion as well as mRNA expression of its parent peptide hormone FNDC5. Simvastatin significantly induced cellular reactive oxygen species levels along with expression of pro- and anti-oxidative genes such as Nox2, and MnSOD and catalase, respectively. Markers of cellular stress such as atrogin-1 mRNA and Bax protein expression were also induced by simvastatin. Decreased cell viability and increased irisin secretion by simvastatin was reversed by antioxidant mito-TEMPO, implying in part that irisin is secreted as a result of increased mitochondrial oxidative stress and subsequent myocyte damage.

Conclusions: Simvastatin increases irisin concentrations in vivo and in vitro. It remains to be determined whether this increase is a result of muscle damage or a protective mechanism against simvastatin-induced cellular stress.

Trial registration: ClinicalTrials.gov NCT00317993 NCT00317993.

Conflict of interest statement

Competing Interests: The parent trial (1) was supported in part by an investigator-initiated grant from MSD Sharp & Dohme (Munich, Germany) and the Wilhelm-Doerenkamp Foundation (Cologne, Germany). Co-author Heiner K. Berthold is a PLOS ONE Editorial Board member. Ioanna Gouni-Berthold has received research grants from 28 Bayer Health Care and honoraria and travel expenses from Genzyme, MSD Sharp & Dohme, Novartis, Novo Nordisk, Pfizer, Ipsen, Bristol Myers Squibb, Amgen, and Otsuka. None of the other authors have relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Irisin and LDL cholesterol concentrations at baseline and after 2 weeks of treatment.

(A) Frequency histogram and normal distribution of irisin baseline concentrations. Data of N = 70 subjects were available. Mean ± SD baseline concentrations were 265±102 ng/ml (range 85 to 518 ng/ml). (B) Percent change from baseline concentrations in LDL cholesterol (left) and irisin (right). (C) Individual changes from baseline to 2-weeks irisin concentrations in the 3 treatment groups. The graph on the right shows the combined data of the subjects receiving simvastatin (either alone or in combination with ezetimibe).

Figure 2. Increased irisin does not correlate with circulating creatine kinase levels.

(A) Serum creatine kinase levels before and 2 weeks after simvastatin treatment. (B) Correlation between change in irisin levels and change in creatine kinase levels in simvastatin-treated subjects. Values are means ± SEM of N = 23 subjects.

Figure 3. Simvastatin increases irisin secretion and FNDC5 mRNA expression in human skeletal muscle cells (HSKMCs).

Measurement of irisin in media (A) and lysate (B) at 24 and 48 hrs after 5 µM simvastatin treatment. FNDC5 mRNA levels were measured by real-time PCR after 5 µM simvastatin treatment for 6 and 24 hrs (C) or 1 and 5 µM for 24 hrs (D). mRNA levels of PGC-1α (E) and atrogin-1 (F) were also measured in simvastatin-treated HSKMCs (5 µM, 6 and 24 hrs). Values are means ± SEM of 4 individual experiments. *Pvs. control.

Figure 4. Simvastatin induces muscle damage, oxidative stress, and apoptosis in human skeletal muscle cells, and mito-TEMPO reverses simvastatin-induced cellular damage and irisin secretion.

(A) Differentiated HSKMCs were incubated with 5 µM simvastatin for 48 and 96 hrs and representative pictures were taken. Enlarged inset pictures are shown for better viewing. (B) The myotube diameter was measured in the images shown in (A) using Image J, as described in the methods. (C) Intracellular oxidative stress levels were measured with DCF-DA 48 hrs after 2 and 10 µM simvastatin treatment. (D–G) Expression of oxidative stress related genes, including Nox2, Nox4, MnSOD, and catalase were measured 24 hrs after 5 µM simvastatin treatment. (H–I) Protein levels of Bcl-2 and Bax after 24 and 48 hrs 5 µM simvastatin treatment and its quantification in HSKMCs. (J) Cell viability was measured by MTT assay after 5 µM simvastatin treatment for 48 hrs. (K) Irisin secretion in media was measured 48 hrs after 5 µM simvastatin treatment. For (J) and (K), 100 µM Mito-TEMPO was administered for 1 hr prior to simvastatin treatment. Values are means ± SEM of 4 individual experiments. RFU: relative fluorescence unit, Statin: simvastatin, TEMPO: mito-TEMPO. *Pvs. control, **P<0.05 vs.48 hr treated (B) or 2 µM simvastatin-treated (C) HSKMCs, †P<0.05 vs. simvastatin-treated HSKMCs.