Ursolic Acid Increases Skeletal Muscle and Brown Fat and Decreases Diet-Induced Obesity, Glucose Intolerance and Fatty Liver Disease

Skeletal muscle Akt activity stimulates muscle growth and imparts resistance to obesity, glucose intolerance and fatty liver disease. We recently found that ursolic acid increases skeletal muscle Akt activity and stimulates muscle growth in non-obese mice. Here, we tested the hypothesis that ursolic acid might increase skeletal muscle Akt activity in a mouse model of diet-induced obesity. We studied mice that consumed a high fat diet lacking or containing ursolic acid. In skeletal muscle, ursolic acid increased Akt activity, as well as downstream mRNAs that promote glucose utilization (hexokinase-II), blood vessel recruitment (Vegfa) and autocrine/paracrine IGF-I signaling (Igf1). As a result, ursolic acid increased skeletal muscle mass, fast and slow muscle fiber size, grip strength and exercise capacity. Interestingly, ursolic acid also increased brown fat, a tissue that shares developmental origins with skeletal muscle. Consistent with increased skeletal muscle and brown fat, ursolic acid increased energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis. These data support a model in which ursolic acid reduces obesity, glucose intolerance and fatty liver disease by increasing skeletal muscle and brown fat, and suggest ursolic acid as a potential therapeutic approach for obesity and obesity-related illness

The Transcription Factor ATF4 Promotes Skeletal Myofiber Atrophy during Fasting

Prolonged fasting alters skeletal muscle gene expression in a manner that promotes myofiber atrophy, but the underlying mechanisms are not fully understood. Here, we examined the potential role of activating transcription factor 4 (ATF4), a transcription factor with an evolutionarily ancient role in the cellular response to starvation. In mouse skeletal muscle, fasting increases the level of ATF4 mRNA. To determine whether increased ATF4 expression was required for myofiber atrophy, we reduced ATF4 expression with an inhibitory RNA targeting ATF4 and found that it reduced myofiber atrophy during fasting. Likewise, reducing the fasting level of ATF4 mRNA with a phosphorylation-resistant form of eukaryotic initiation factor 2α decreased myofiber atrophy. To determine whether ATF4 was sufficient to reduce myofiber size, we overexpressed ATF4 and found that it reduced myofiber size in the absence of fasting. In contrast, a transcriptionally inactive ATF4 construct did not reduce myofiber size, suggesting a requirement for ATF4-mediated transcriptional regulation. To begin to determine the mechanism of ATF4-mediated myofiber atrophy, we compared the effects of fasting and ATF4 overexpression on global skeletal muscle mRNA expression. Interestingly, expression of ATF4 increased a small subset of five fasting-responsive mRNAs, including four of the 15 mRNAs most highly induced by fasting. These five mRNAs encode proteins previously implicated in growth suppression (p21Cip1/Waf1, GADD45α, and PW1/Peg3) or titin-based stress signaling [muscle LIM protein (MLP) and cardiac ankyrin repeat protein (CARP)]. Taken together, these data identify ATF4 as a novel mediator of skeletal myofiber atrophy during starvation

Ursolic acid reduces diet-induced fatty liver disease.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing ursolic acid (UA) for 6 weeks. UA concentrations were 0.27% (B–C and E) or 0.14% (A, D and F–G). Data are means ± SEM. *P<0.05 by t-test. (A) Liver weights. n≥12 mice per diet. (B) Liver H&E-stained sections. 20x magnification. (C) Liver osmium-stained sections, 10x magnification. (D) Hepatic triglyceride content. n = 5 mice per diet. (E) Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. n = 5 mice per diet. (F) Liver mRNA levels were determined using qPCR. Levels in UA-treated mice were normalized to the average levels in mice fed HFD lacking ursolic acid, which were set at 1. n = 10 mice per diet. (G) Livers were harvested and subjected to SDS-PAGE and immunoblot analysis with anti-ACC and anti-tubulin antibodies. Upper: representative immunoblots. Lower: ACC and tubulin levels were quantitated with densitometry. In each mouse, the ACC/tubulin ratio was normalized to the average ACC/tubulin ratio in mice fed HFD lacking ursolic acid. n = 6 mice per diet.</p

Chronic, but not acute, ursolic acid treatment increases food intake and energy expenditure.

<p>Mice were fed high fat diet (HFD) lacking or containing 0.27% ursolic acid (UA) for either 3 days (acute treatment) or 6 weeks (chronic treatment), and then food intake (A) and energy expenditure (B) were determined using a comprehensive lab animal monitoring system (CLAMS). <i>Left panels</i>: hourly measurements. Data are means from 12 mice per diet. <i>Right panels</i>: cumulative measurements during the dark and light periods. Data are means ± SEM from 12 mice per diet. <i>P-</i>values were determined with unpaired t-tests. *<i>P</i><0.05.</p

Ursolic acid increases exercise capacity, does not alter blood pressure, and reduces resting heart rate in high fat-fed mice.

<p>Mice were fed high fat diet (HFD) lacking or containing 0.27% ursolic acid (UA) for 17 weeks, and then exercise treadmill capacity was determined according to an established protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039332#pone.0039332-Arany1" target="_blank">[36]</a> (A) and resting blood pressure and heart rate were determined with tail cuff plethysmography (B and C). Data are means ± SEM from ≥7 mice per diet. P-values were determined with t-tests. *P<0.05.</p

Ursolic acid reduces diet-induced obesity and glucose intolerance.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing 0.14% ursolic acid (UA) for 6 weeks. Data are means ± SEM. *P<0.05 by t-test. (A) Total body weight was measured at the indicated times. n≥12 mice per diet. (B) Weights of bilateral epididymal and retroperitoneal fat pads. n = 10 mice per diet. (C) Fasting blood glucose levels. Mice were fasted for 16 h prior to tail vein glucose measurements. n≥12 mice per diet. (D) Glucose tolerance tests. Following a 16 h fast, 1 g/kg glucose was administered by i.p. injection at time  = 0 min. Blood glucose was then measured via the tail vein at the indicated times. n = 10 mice per diet. Left, blood glucose values. Right, areas under the curves.</p

In mice fed a high fat diet, ursolic acid increases skeletal muscle Akt signaling, anabolic mRNA expression, grip strength, skeletal muscle mass, and fast and slow skeletal muscle fiber size.

<p>Mice were provided ad libitum access to high fat diet (HFD) lacking or containing 0.14% ursolic acid (UA) for 6 weeks. Data are means ± SEM. *<i>P</i><0.05 by t-test. (A) Triceps muscles here harvested and subjected to SDS-PAGE and immunoblot analysis with anti-phospho(Ser473)-Akt and anti-Akt antibodies. <i>Upper</i>: representative immunoblots. <i>Lower</i>: Phospho-Akt (P-Akt) and total Akt levels were quantitated with densitometry. In each mouse, the phospho-Akt/total Akt ratio was normalized to the average phospho-Akt/total Akt ratio in mice fed HFD lacking UA. n = 5 mice per diet. (B) Quadriceps mRNA levels were determined using qualitative real-time RT-PCR (qPCR). Levels in UA-treated mice were normalized to the average levels in mice fed HFD lacking ursolic acid, which were set at 1. n = 10 mice per diet. (C) Grip strength. n = 10 mice per diet. (D) Weights of bilateral quadriceps and triceps brachii (triceps). n≥12 mice per diet. (E) Slow and fast muscle fiber diameters. Sections of triceps muscle were subjected to immunohistochemical analysis with anti-slow myosin and anti-fast myosin antibodies, and then fiber diameter was measured. Slow fibers: n≥50 fibers/triceps from 5 mice per condition. Fast fibers: n≥100 fibers/triceps from 5 triceps per condition.</p