Pgc1

1. ND HFD p=0.24 p=0.79 Pgc1 ND HFD Figure S1. Effect of a 12-week high-fat diet on mRNA expression of genes involved in fatty acid oxidation and mitochondrial biogenesis in epididymalfat. Expression of the long-chain acyl-CoAdehydrogenase (Acadl) gene, encoding an enzyme catalyzing the first step of fatty acid oxidation, and of the peroxisomeproliferator-activated receptor-gamma coactivator-1 (Pgc-1) gene, which encodes a transcription factor coordinating the expression of 16 nuclear and mitochondrial genes involved in mitochondrial biogenesis, were examined with quantitative RT-PCR. Means ± SEM are presented for ND (n=5-6) and HFD (n=8) rats. Data were analyzed with Student t-test.

2. Interscapular brown fat Retroperitoneal fat ND HFD 1 2 3 4 5 1 2 3 4 5 6 7 8 1 A 1 2 3 4 5 1 2 3 4 5 6 7 8 1 B 64 kDa 64 kDa 49 kDa 49 kDa 37 kDa 37 kDa UCP-1 Figure S2. Western blot analysis of UCP-1 in retroperitoneal fat of normal diet (ND) or high-fat diet (HFD) animals. A. UCP1 expression of protein extracts (10 μg total protein) from retroperitoneal fat pads of animals on a normal diet (ND) or high-fat diet (HFD). Positive control was protein extract (0.5 μg) from interscapular brown fat. Inter-individual variability in the quantity of UCP1 protein observed in HFD animals is likely due a sampling issue, i.e. brown adipocytes occur in patches that are not uniformly distributed across retroperitoneal fat pads; therefore, it is possible that not all tissue sections from HFD animals analyzed in this experiment contained brown fat. B. Ponsceau S stain of the immunoblot (A) confirming equal loading of total protein. Note that, in addition to loading 0.5 μg of total protein from interscapular brown fat, a molecular weight, pre-stained ladder was loaded as seen in the ponsceau S stain (B). 26 kDa 26 kDa

3. Ucp1 NC Retro Epi Liver SM Figure S3. PCR of the uncoupling protein 1 gene (Ucp1) in retroperitoneal and epididymal fat, soleus muscle and liver. The results show that Ucp1 is expressed in retroperitoneal fat (Retro) but not in epididymal fat (Epi), soleus muscle (SM) and liver of high-fat diet (HFD) animals (pooled cDNA, 8 animals). Negative control (NC) was water.

4. ND HFD SM NC Figure S4. PCR of the myogenic factor 5 gene (Myf5) in retroperitoneal fat and soleus muscle. The results show that Myf5 is not expressed in retroperitoneal fat of normal diet (ND) or high-fat diet (HFD) animals (pooled cDNA, ND: 5 animals, HFD: 8 animals) but it is expressed in soleus muscle (SM). Negative control (NC) was water.

5. 20x magnification – UCP1 (left), Negative control (right) 40x magnification – UCP1 (left), Negative control (right) Figure S5. UCP1 immunohistochemistry of retroperitoneal fat and its negative controls. Sections of retroperitoneal fat were immunostained with rabbit anti-UCP1 (1:500, U6382, Sigma, St Louis, MO, USA) using the VECTASTAIN Elite ABC Kit and the VECTOR DAB Substrate Kit for Peroxidase (Vector Laboratories, Burlingame, CA, USA) and again examined with light microscopy (Eclipse, TE-2000-S, Nikon UK Limited, Kingston upon Thames, UK) under 20x and 40x magnifications. Negative controls were performed with the same protocol, with the primary antibody being omitted.