Lipid droplet-associated proteins in high-fat fed mice with the effects of voluntary running and diet change  Rita Rinnankoski-Tuikka, Juha J. Hulmi, Sira Torvinen, Mika Silvennoinen, Maarit Lehti, Riikka Kivelä, Hilkka Reunanen, Urho M. Kujala, Heikki Kainulainen  Metabolism - Clinical and Experimental  Volume 63, Issue 8, Pages 1031-1040 (August 2014) DOI: 10.1016/j.metabol.2014.05.010 Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 1 Summary of study set up. Graph summarizing the experiment set up and data collection points. During a 19-week experiment, C57BL/6J mice were studied in six groups: low-fat diet sedentary, low-fat diet active, high-fat diet sedentary, high-fat diet active and two groups, which were high-fat sedentary for nine weeks, after which divided into low-fat sedentary or low-fat active groups. Group size varied from 14 to 15 animals. LFS=low-fat sedentary, LFA=low-fat active, HFS=high-fat sedentary, HFA=high-fat active, DCS=diet change sedentary, DCA=diet change active. Metabolism - Clinical and Experimental 2014 63, 1031-1040DOI: (10.1016/j.metabol.2014.05.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 2 Weekly running distance of active mice. The graph of weekly running distance (km) demonstrates increasing running distances during the first weeks, but declining thereafter until the end of the study in all groups. On the ninth week and from the twelfth week onwards, the LF mice ran significantly longer distances per week than HF mice. The colored areas indicate ±SD: LFA=black, HFA=light gray, DCA=middle gray. ⁎P<0.05 LFA vs. HFA. LFA=low-fat active, HFA=high-fat active, DCA=diet change active. Metabolism - Clinical and Experimental 2014 63, 1031-1040DOI: (10.1016/j.metabol.2014.05.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 3 Electron microscopic images. Representative electron microscopic images from the soleus muscle in LFS (A), LFA (B), HFS (C), HFA (D), DCS (E) and in DCA (F). In contrast to LFS, in HFS there are many intramyocellular lipid droplets (arrow), which seem to be often located in close proximity to mitochondria (M). The densely packed areas of subsarcolemmal mitochondria (SS) are often adjacent to capillaries (C). The results from micrograph quantification on the amount of lipids can be seen in Table 3. Scale bar 2μm. LFS=low-fat sedentary, LFA=low-fat active, HFS=high-fat sedentary, HFA=high-fat active, DCS=diet change sedentary, DCA=diet change active. Metabolism - Clinical and Experimental 2014 63, 1031-1040DOI: (10.1016/j.metabol.2014.05.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 4 Protein and mRNA expressions of PLIN5, PLIN2 and PLIN3. Protein expression of PLIN5 (A) was significantly higher in HF, when compared to their LF and DC counterparts. The mRNA expression levels of PLIN5 were very similar to the protein expression pattern (B). HFA and DCA mice had higher PLIN2 (C) protein expression levels than LFS mice. The mRNA expression level of PLIN2 (D) in HF, especially in active HF and DC, was significantly higher than in LF mice. No significant changes in PLIN3 (E) protein expression level were observed. The PLIN3 (F) mRNA expression was significantly up-regulated in the active LF and HF groups than respective sedentary groups. There were no differences between the sedentary groups. Representative Western blot images (G). The protein expression results are normalized to the mean value of LFS and presented as relative to Ponceau S, whereas the mRNA gene expressions are normalized to the expression of GAPDH and the results expressed in relation to the LFS mean. Values are mean±SD. AU=arbitrary units. LFS=low-fat sedentary, HFS=high-fat sedentary, DCS=diet change sedentary, LFA=low-fat active, HFA=high-fat active, DCA=diet change active. ⁎P<0.05 vs. LFS, †P<0.05 vs. LFA, ‡P<0.05 vs. HFS, §P<0.05 vs. DCA. Metabolism - Clinical and Experimental 2014 63, 1031-1040DOI: (10.1016/j.metabol.2014.05.010) Copyright © 2014 Elsevier Inc. Terms and Conditions