The obesogenic effects of polyunsaturated fatty acids are dependent ...

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Plasma analysis. Insulin, glucose (16) and lipid metabolites (35) were measured in plasma as earlier
described.
Tissue lipid analysis. Total lipids were extracted from diets and liver with chloroform: methanol,
2:1 (v/v). Lipid classes were analyzed in liver samples using an automated High-Performance Thin
Layer Chromatography (HPTLC) system (Camaq, Switzerland) and separated on HPTLC plates
coated with silica gel 60 F (35). Fatty acid composition of total lipids from diets was analyzed on a
capillary gas chromatograph with flame ionization detector (Perkin Elmer, USA) (36).
Histology. Sections of adipose tissue were fixed, dehydrated, embedded in paraffin blocks, cut into
3 μm thick sections and stained with eosin and hematoxylin as described (37). Sections were
visually examined using an Olympus BX 51 binocular microscope (Olympus Corporation, Japan),
fitted with an Olympus DP50 3.0 camera.
RT-qPCR. Total RNA was purified from mouse tissue using Trizol (Invitrogen). Reverse
transcription (RT) was performed and cDNA was analyzed in duplicates by qPCR using the ABI
PRISM 7700 Sequence Detection System (Applied Biosystems) as earlier described (38). Primers
for RT-qPCR were designed using Primer Express 2.0 (Applied Biosystems) and are available on
request.
Statistics. Data represent mean ± SEM. ANOVA, post hoc pairwise comparison: Student t-test (RTqPCR
analysis) or Tukey HSD test (GTT, ITT and organ weights). Newman-Keuls test
(nonparametric due to non-homogenous variances, remaining data sets. A value of P