Statin Treatment Increases Lifespan and Improves Cardiac Health in Drosophila by Decreasing Specific Protein Prenylation

Statins such as simvastatin are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and standard therapy for the prevention and treatment of cardiovascular diseases in mammals. Here we show that simvastatin significantly increased the mean and maximum lifespan of Drosophila melanogaster (Drosophila) and enhanced cardiac function in aging flies by significantly reducing heart arrhythmias and increasing the contraction proportion of the contraction/relaxation cycle. These results appeared independent of internal changes in ubiquinone or juvenile hormone levels. Rather, they appeared to involve decreased protein prenylation. Simvastatin decreased the membrane association (prenylation) of specific small Ras GTPases in mice. Both farnesyl (L744832) and type 1 geranylgeranyl transferase (GGTI-298) inhibitors increased Drosophila lifespan. These data are the most direct evidence to date that decreased protein prenylation can increase cardiac health and lifespan in any metazoan species, and may explain the pleiotropic (non-cholesterol related) health effects of statins

Food consumption as measured by FPA does not change in response to simvastatin or methoprene.

a<p>These data are the means plus or minus the standard error of the plaque number per cm² per fly per hour. The plaque values are not significantly different as judged by ANOVA followed by Tukey's multiple comparison test. See <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039581#s4" target="_blank">Materials and Methods</a></i> for specifics regarding the studies.</p

Locomotor activity of L744832 and GGTI-298 treated <i>Drosophila</i>.

1<p>Groups of 10 newly eclosed Drosophila were maintained for 14 days at 25°C with medium containing either an equal volume of vehicle, 20 µM L744832, or 300 µM GGTI-298 in a Drosophila Activity Monitoring System.</p

Ubiquinone administration slightly reduces lifespan.

<p>Shown are the lifespan of flies consuming food containing no drugs [control (○)]; 240 µM simvastatin (□); 5 mM CoQ10 (▾); 5 mM CoQ10 and 240 µM simvastatin (•). The mean lifespans of the flies treated with simvastatin alone (P<0.0001) and with 5 mM CoQ10 plus simvastatin (P = 0.0058) were significantly greater than that of control. The mean lifespan of the flies treated with 5 mM CoQ10 alone was significantly less than control (P = 0.0001)</p

Inhibition of farnesyl transferase or geranylgeranyl transferase I activity increases <i>Drosophila</i> lifespan.

<p>Shown in panel A are the lifespans of <i>Drosophila</i> in the absence of drugs [control (○)]; and in the presence of 5 µM (□); 10 µM (▵); or 20 µM (▾) L744832. The mean lifespan of flies treated with 5, 10, and 20 µM L744832 were significantly increased relative to control (P = 0.0089, P<0.0001, and P = 0.0019, respectively). In panel B are shown the lifespans of <i>Drosophila</i> in the absence of drugs [control (○)]; and in the presence of 100 µM (▵) or 300 µM (▾) GGTI-298. The mean lifespan of flies treated with 100 and 300 µM GGTI-298 was significantly increased relative to control (P<0.0001 and P<0.0001, respectively).</p

Locomotor activity of simvastatin treated <i>Drosophila</i>

1<p>Groups of 10 newly eclosed Drosophila were maintained for 14 days at 25°C with medium containing either an equal volume of vehicle or 2.4 mM simvastatin in a TriKinetics Drosophila Activity Monitoring System.</p>2<p>Infrared beam disruptions per 24 hours.</p

Simvastatin administration to mice decreased prenylation of specific small G proteins, as measured by a decrease in their membrane association.

<p>Panel A, Western blot showing the level of Rab4, Ras, calnexin, and α-tubulin in the cytoplasmic and membrane fractions purified from the liver of control and simvastatin treated mice. Panel B shows the quantification of the data shown in Panel A. The levels of Rab4 in the cytoplasmic fractions were normalized to the level of α-tubulin in each sample. The levels of Rab 4 in the membrane fractions were normalized to the level of calnexin in each sample. Means and standard errors obtained with tissue from control (open bars) and simvastatin treated (closed bars) mice are shown. One of the precipitated membrane samples was overloaded on this blot, and the amount of Rab4 reported in panel B was determined from another blot. The scanner units were adjusted to facilitate comparisons between membrane and cytoplasmic fractions.</p

Food consumption does not change in response to chemical additions to the diet as determined by CAFE assays analyzed with Tukey's Multiple Comparison Test^a.

a<p>See <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039581#s4" target="_blank">Materials and Methods</a></i> for specifics regarding the studies. The fly medium contained either an equal volume of vehicle, 2.4 mM simvastatin, 320 µM methoprene, or their combination.</p>b<p>Mean difference between food consumption in the absence and presence of the drug(s).</p>c<p>The notation ** indicates the difference was highly significant (P≤0.01). The notation NS indicates that the results were not significantly different.</p

The acetyl CoA-mevalonate pathway in insects and mammals.

<p>The X at the head of the cholesterol branch of the pathway indicates that insects lack several enzymes required for cholesterol biosynthesis.</p

Fly weight in response to drug treatments.

1<p>Newly eclosed Drosophila were maintained for 12 days at 25°C with medium containing either an equal volume of vehicle, 2.4 mM simvastatin, 320 µM methoprene, 20 µM L744832, or 300 µM GGTI-298. Groups of 10 flies were anesthetized with CO₂, immediately removed from the flybottles, and weighed (n = 12).</p>2<p>Column values with the same superscript letters are not significantly different, as determined by one way ANOVA. See <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039581#s4" target="_blank">Materials and Methods</a></i> for specifics regarding the studies.</p