Effects of Lipid-Lowering Drugs on Irisin in Human Subjects In Vivo and in Human Skeletal Muscle Cells Ex Vivo

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

Effects of lipid-lowering drugs on high-density lipoprotein subclasses in healthy men-a randomized trial.

Investigating the effects of lipid-lowering drugs on HDL subclasses has shown ambiguous results. This study assessed the effects of ezetimibe, simvastatin, and their combination on HDL subclass distribution.A single-center randomized parallel 3-group open-label study was performed in 72 healthy men free of cardiovascular disease with a baseline LDL-cholesterol of 111±30 mg/dl (2.9±0.8 mmol/l) and a baseline HDL-cholesterol of 64±15 mg/dl (1.7±0.4 mmol/l). They were treated with ezetimibe (10 mg/day, n = 24), simvastatin (40 mg/day, n = 24) or their combination (n = 24) for 14 days. Blood was drawn before and after the treatment period. HDL subclasses were determined using polyacrylamide gel-tube electrophoresis. Multivariate regression models were used to determine the influence of treatment and covariates on changes in HDL subclass composition.Baseline HDL subclasses consisted of 33±10% large, 48±6% intermediate and 19±8% small HDL. After adjusting for baseline HDL subclass distribution, body mass index, LDL-C and the ratio triglycerides/HDL-C, there was a significant increase in large HDL by about 3.9 percentage points (P<0.05) and a decrease in intermediate HDL by about 3.5 percentage points (P<0.01) in both simvastatin-containing treatment arms in comparison to ezetimibe. The parameters obtained after additional adjustment for the decrease in LDL-C indicated that about one third to one half of these effects could be explained by the extent of LDL-C-lowering.In healthy men, treatment with simvastatin leads to favorable effects on HDL subclass composition, which was not be observed with ezetimibe. Part of these differential effects may be due to the stronger LDL-C-lowering effects of simvastatin.ClinicalTrials.gov NCT00317993

HDL subclass distribution before (solid line) and after 2 weeks of treatment (dotted lines).

<p>The data are means (95% confidence intervals) of the percentage of total HDL cholesterol. (A) ezetimibe, (B) simvastatin, (C) ezetimibe plus simvastatin.</p

Demographic data and biochemical baseline characteristics of the study participants.

<p>BMI, body mass index; BIA, bioelectrical impedance analysis; LDL, low-density lipoprotein; HDL, high-density lipoprotein.</p><p>Data are presented as mean ± SD or counts (percentages). There were no significant differences between the 3 treatment groups at baseline. Large HDL are composed of subclasses 1–3, intermediate HDL of subclasses 4–7 and small HDL of subclasses 8–10.</p>†<p>percent of total HDL cholesterol.</p><p>*HDL composition data were not available in 2 subjects in the ezetimibe group.</p

CONSORT flow diagram: Flow of participants through the trial.

<p>CONSORT flow diagram: Flow of participants through the trial.</p

Plasma lipoprotein concentrations before and after treatment.

<p>Data are presented as mean ± SD and in the case of triglycerides as medians (interquartile range). Each group comprised n = 24 subjects (HDL subclass data in the ezetimibe group were available in only n = 22 subjects).</p

Multivariate linear regression models.

<p>In <i>model 1</i>, only treatment group and the respective baseline HDL subclass composition was modelled. In <i>model 2</i>, BMI, baseline LDL cholesterol and the baseline ratio of triglycerides to HDL cholesterol were used as additional explanatory variables. In <i>model 3</i>, the change in LDL cholesterol was added as explanatory variable and the specific drug treatments were added as dummy variables.</p><p><i>Note:</i> for ease of interpretation, coefficients for change in LDL cholesterol are given per decrease by 20% (corresponding to approximately the change in the ezetimibe group or one half of the change in the simvastatin group or one third of the change in the combination treatment group).</p

HDL subclasses baseline data and correlation with demographic and biochemical parameters.

<p>The data are Pearson correlation coefficients. There were no significant correlations with (data not shown) estimated glomerular filtration rate, thyroid function tests, cholesterol synthesis and absorption markers, and smoking status.</p><p>HOMA, homeostasis model assessment; hsCRP, high-sensitivity C-reactive protein; PCSK9, proprotein convertase subtilisin/kexin type 9.</p

Mean (95% confidence intervals) proportions of HDL subclasses before and 2 weeks after treatment.

<p>(A) large HDL, (B) intermediate HDL, (C) small HDL.</p

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

<p>(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. *P<0.05 <i>vs.</i> control, **P<0.05 <i>vs.</i>48 hr treated (B) or 2 µM simvastatin-treated (C) HSKMCs, †P<0.05 <i>vs.</i> simvastatin-treated HSKMCs.</p