Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease

Abstract

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.

Conflict of interest statement

Competing Interests: The University of Iowa Research Foundation has applied for patents related to this work (WO/2011/146768/A1, /PCT/US2011/037238, Methods for Inhibiting Muscle Atrophy). The authors also declare that CMA is a co-founder and officer of Emmyon, Inc. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. 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.

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) Triceps muscles here harvested and subjected to SDS-PAGE and immunoblot analysis with anti-phospho(Ser473)-Akt and anti-Akt antibodies. Upper: representative immunoblots. Lower: 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.

Figure 2. Ursolic acid increases exercise capacity, does not alter blood pressure, and reduces resting heart rate in high fat-fed mice.

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) 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

Figure 3. Ursolic acid reduces diet-induced obesity and glucose intolerance.

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

Figure 4. Ursolic acid reduces diet-induced fatty liver disease.

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

Figure 5. Ursolic acid increases interscapular brown fat.

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) Interscapular fat pads were harvested and dissected into white fat and brown fat components, which were then weighed. n = 10 mice per diet. (B) Protein from the entire interscapular fat pad was isolated and subjected to SDS-PAGE and immunoblot analysis with anti-UCP1 and anti-actin antibodies. Upper: representative immunoblots. Lower: UCP1 and actin levels were quantitated with densitometry. In each mouse, the UCP1/actin ratio was normalized to the average UCP1/actin ratio in mice fed HFD lacking UA, which was set at 1. n = 8 mice per diet. (C) Cold tolerance test. Following 6 weeks of HFD ± UA, a baseline rectal temperature was obtained at 21°C (t = 0 hours). Mice were then moved to 4°C, where rectal temperature was measured hourly. n = 8 mice per diet.

Figure 6. Chronic, but not acute, ursolic acid treatment increases food intake and energy expenditure.

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). Left panels: hourly measurements. Data are means from 12 mice per diet. Right panels: cumulative measurements during the dark and light periods. Data are means ± SEM from 12 mice per diet. P-values were determined with unpaired t-tests. *P<0.05.